Treatment of sarcoidosis using placental stem cells

ABSTRACT

Provided herein are compositions and methods of treating individuals having sarcoidosis or a sarcoidosis-related disease or disorder, using placental cells, e.g., the placental stem cells and placental multipotent cells (PDACs) described herein. Also provided herein are kits comprising said cells or a composition thereof.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/322,272, filed Apr. 8, 2010, which isincorporated by reference herein in its entirety.

1. FIELD

Provided herein are isolated placental cells (e.g., placental stemcells), populations of the placental stem cells, and methods of usingthe same to treat sarcoidosis or a sarcoidosis-related disease, disorderor condition, for example, systemic sarcoidosis, cutaneous sarcoidosis,Löfgren's syndrome, neurosarcoidosis, pulmonary sarcoidosis, cardiacsarcoidosis, ocular sarcoidosis, or sarcoidosis with the involvement ofother organs or tissues.

2. BACKGROUND

Sarcoidosis, also called sarcoid disease or Besnier-Boeck disease, is amultisystem granulomatous inflammatory disease that can affect a varietyof organs (e.g., lungs, skin, heart, eyes, liver, nervous system,kidneys, or musculoskeletal system). The etiology of sarcoidosis hasremained unknown, though it has been linked to alterations in the immuneresponse after exposure to an environmental, occupational, or infectiousagent in genetically susceptible individuals.

Steroid therapy (e.g., corticosteroids such as prednisone) has been thestandard treatment for sarcoidosis. However, the use of corticosteroidshas a number of drawbacks. For example, certain patients do not respondto steroid therapy. In addition, corticosteroids have several seriousside effects, and their use is typically limited to progressive orsevere conditions. Accordingly, there exists a need for new methods oftreating sarcoidosis, using, for example, human placental stem cells.

3. SUMMARY

In one aspect, provided herein are methods of treating, managing,ameliorating or preventing sarcoidosis, or one or moresarcoidosis-related diseases, disorders and/or conditions, e.g.,associated with, resulting in or caused by sarcoidosis. Also providedherein are kits for use in the treatment of sarcoidosis, or a disease,disorder or condition caused by, or relating to, sarcoidosis.

In one embodiment, provided herein is a method of treating an individualhaving sarcoidosis, or a sarcoidosis-related disease, disorder orcondition, comprising administering to the individual a therapeuticallyeffective amount of tissue culture plastic-adherent placental cells,e.g., placental multipotent cells or placental stem cells, also referredto herein as PDACs (placenta derived adherent cells, e.g., theplacenta-derived adherent cells described in Section 4.2, below), orculture medium conditioned by PDACs (e.g., placental stem cells), in anamount and for a time sufficient for detectable improvement of one ormore symptoms of sarcoidosis, or said disease, disorder or condition.

In another aspect, provided herein is the use of placental cells (e.g.,placental stem cells) in the manufacture of a medicament for treating,managing, ameliorating or preventing sarcoidosis or diseases, disordersand/or conditions caused by, or relating to, sarcoidosis.

In another embodiment, provided herein are kits comprising PDACs or atherapeutic cell composition thereof, which can be prepared in apharmaceutically acceptable form, for example by mixing with apharmaceutically acceptable carrier, and an applicator, along withinstructions for use.

In certain embodiments, the method comprises administering placentalcells (e.g., placental stem cells) to said individual in an amount andfor a time sufficient for detectable improvement of one or more symptomsof said sarcoidosis or said disease or disorder, as compared to theindividual prior to administration of said placental stem cells.

In certain embodiments, said therapeutically effective amount ofplacental cells reduces, e.g., detectably reduces, the number of, ordegree of severity of, or reduces the rate of increase in the number of,or degree of severity, said one or more symptoms in said individual,e.g., and/or detectable improvement, of one or more symptoms ofsarcoidosis or sarcoidosis-related diseases, disorders or conditions. Inspecific embodiments, said therapeutically effective amount of placentalcells reduces, e.g., detectably reduces, the number of, or degree ofseverity of, or reduces the rate of increase in the number of, or degreeof severity, one or more granulomas in said individual. In oneembodiment, said therapeutically effective amount is an amount thatresults in a detectable improvement in at least one symptom of saidsarcoidosis.

In one specific embodiment, said sarcoidosis is systemic sarcoidosis,and said symptoms comprise one ore more of formation of granulomas,fatigue, weight loss, fever, aches, pains, arthritis, dry eyes, swellingof the knees, blurry vision, shortness of breath, cough or skin lesions.

In another specific embodiment, said sarcoidosis is cutaneoussarcoidosis, and said symptoms comprise a skin lesion. In a morespecific embodiment, said cutaneous sarcoidosis comprises one or more ofannular sarcoidosis, erythrodermic sarcoidosis, ichthyosiformsarcoidosis, hypopigmented sarcoidosis, morpheaform sarcoidosis, mucosalsarcoidosis, papular sarcoid, scar sarcoid, subcutaneous sarcoidosis orulcerative sarcoidosis. In a more specific embodiment, said skin lesioncomprises one or more of erythema nodosum, nummular eczema, erythemamultiforme, calcinosis cutis and pruritus, lupus pernio, skin plaques,maculopapular eruptions, nodular lesions deeper in the skin orinfiltration of old scars.

In another specific embodiment, said sarcoidosis is Löfgren's syndrome,and said symptoms comprise one or more of erythema nodosum, bilateralhilar denopathy, arthritis, arthralgias or fever. In another specificembodiment, said sarcoidosis is neurosarcoidosis, and said symptomscomprise one or more of formation of granulomas in the nervous system,headache, confusion, malaise or facial paralysis. In another specificembodiment, said sarcoidosis is pulmonary sarcoidosis, and said symptomscomprise one or more of cough, shortness of breath, chest pain, afeeling of tightness in the chest, hoarseness, nasal obstruction andrecurrent or persistent sinusitis. In another specific embodiment, saidsarcoidosis is cardiac sarcoidosis, and said symptoms comprise one ormore of chest pain, palpitations, congestive heart failure, pericarditisor papillary muscle dysfunction, shortness of breath, ankle swelling,irregular heart beat or sudden death. In another specific embodiment,said sarcoidosis is ocular sarcoidosis, and said symptoms comprise oneor more of red or watery eyes, granulomatous uvetis, iris nodules,retinochoroiditis, conjunctivitis, lacrimal gland involvement orproptosis. In another specific embodiment, said sarcoidosis issarcoidosis with the involvement of other organs or tissues, e.g.,sarcoidosis with musculoskeletal, hepatic, hematologic, psychiatric,renal, splenic, nasal sinus, oral, gastric or intestinal, endocrine,pleural or reproductive involvement. In another specific embodiment,said sarcoidosis is sarcoidosis of one or more organs or tissues otherthan pulmonary tissues or organs. In a more specific embodiment, saidsarcoidosis is sarcoidosis of one or more organs or tissues other thanthe lungs.

In certain embodiments, the method of treatment comprises administeringat least about 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, or 1×10¹⁰placental cells to said individual. In another specific embodiment, saidplacental cells have been proliferated in vitro for no more than 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 population doublings. In anotherembodiment of any of the embodiments herein, said placental cells havebeen cryopreserved and thawed prior to said administering.

In certain embodiments of any of the above methods, said placental cellsare adherent to tissue culture plastic and are CD34⁻, CD10⁺, CD105⁺ andCD200⁺ as detected by flow cytometry. In a specific embodiment, theplacental cells have the capacity to differentiate into osteogenic orchondrogenic cells. In another embodiment, said placental cells areadherent to tissue culture plastic; CD34⁻, CD10⁺, CD105⁺ and CD200⁺ asdetected by flow cytometry; and have the capacity to differentiate intocells having one or more characteristics of osteogenic or chondrogeniccells, e.g., characteristics of osteocytes or chondrocytes. In otherembodiments, the placental cells additionally have the ability todifferentiate into cells having one or more characteristics of neuralcells or neurogenic cells, e.g., characteristics of neurons; one or morecharacteristics of glial cells, e.g., characteristics of glia orastrocytes; one or more characteristics of adipocytic cells, e.g.,characteristics of adipocytes; one or more characteristics of pancreaticcells; and/or one or more characteristics of cardiac cells.

In another embodiment, said placental cells are CD34⁻, CD10⁺, CD105⁺ andCD200⁺, and one or more of CD38⁻, CD45⁻, CD80⁻, CD86⁻, CD133⁻,HLA-DR,DP,DQ⁻, SSEA3⁻, SSEA4⁻, CD29⁺, CD44⁺, CD73⁺, CD90⁺, CD105⁺,HLA-A,B,C⁺, PDL1⁺, ABC-p⁺, and/or OCT-4⁺, as detected by flow cytometryand/or RT-PCR. In another embodiment, said placental cells are CD34⁻,CD45⁻, CD10⁺, CD90⁺, CD105⁺ and CD200⁺, as detected by flow cytometry.In another embodiment, said placental cells are CD34⁻, CD45⁻, CD10⁺,CD80⁻, CD86⁻, CD90⁺, CD105⁺ and CD200⁺, as detected by flow cytometry.In another embodiment, said placental cells are CD34⁻, CD45⁻, CD10⁺,CD80⁻, CD86⁻, CD90⁺, CD105⁺ and CD200⁺, and additionally one or more ofCD29⁺, CD38⁻, CD44⁺, CD54⁺, SH3⁺ or SH4⁺, as detected by flow cytometry.In another embodiment, said placental cells are CD34⁻, CD38⁻, CD45⁻,CD10⁺, CD29⁺, CD44⁺, CD54⁺, CD73⁺, CD80⁻, CD86⁻, CD90⁺, CD105⁺, andCD200⁺ as detected by flow cytometry.

In another embodiment, said CD34⁻, CD10⁺, CD105⁺ and CD200⁺ placentalcells are additionally one or more of CD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻),HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, or Programmed Death-1 Ligand (PDL1)⁺, or anycombination thereof. In another specific embodiment, said placentalcells are CD34⁻, CD38⁻, CD45⁻, CD10⁺, CD29% CD44⁺, CD54⁺, CD73⁺, CD80⁻,CD86⁻, CD90⁺, CD105⁺, CD117⁻, CD133⁻, CD200⁺, KDR⁻ (VEGFR2⁻),HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, or Programmed Death-1 Ligand (PDL1)⁺, asdetected by flow cytometry.

In another embodiment, any of the placental cells described herein areadditionally ABC-p⁺, as detected by flow cytometry, or OCT-4⁺ (POU5F1⁺),e.g., as determined by RT-PCR, wherein ABC-p is a placenta-specific ABCtransporter protein (also known as breast cancer resistance protein(BCRP) and as mitoxantrone resistance protein (MXR)). In anotherembodiment, any of the placental cells described herein are additionallySSEA3⁻ or SSEA4⁻, e.g., as determined by flow cytometry, wherein SSEA3is Stage Specific Embryonic Antigen 3, and SSEA4 is Stage SpecificEmbryonic Antigen 4. In another embodiment, any of the placental cellsdescribed herein are additionally SSEA3⁻ and SSEA4⁻.

In another embodiment of the methods described herein, any of theplacental cells described herein are additionally one or more of MHC-I⁺(e.g., HLA-A,B,C⁺), MHC-II⁻ (e.g., HLA-DP,DQ,DR⁻) or HLA-G⁻. In anotherembodiment, any of the placental cells described herein are additionallyeach of MHC-I⁺ (e.g., HLA-A,B,C⁺), MHC-II⁻ (e.g., HLA-DP,DQ,DR⁻) andHLA-G⁻, as detected by flow cytometry.

In another embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ cells areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, CD80⁻, CD86⁻,SH3⁺ or SH4⁺. In another embodiment, the cells are additionally CD44⁺.In another embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cellsare additionally one or more of CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺,CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻,CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR⁴⁻, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻,ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, or ProgrammedDeath-1 Ligand (PDL1)⁺, or any combination thereof. In anotherembodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells areadditionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54/ICAM⁺,CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺,SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻, CD144/VE-cadherin^(dim),CD184/CXCR⁴⁻, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻),HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, and Programmed Death-1 Ligand(PDL1)⁺, as detected by flow cytometry.

In other embodiments of the methods disclosed herein, the isolatedplacental cells are CD200⁺ and HLA-G⁻; CD73⁺, CD105⁺, and CD200⁺; CD200⁺and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁻; CD73⁺ and CD105⁺; or OCT-4⁺; orany combination thereof, as detected by flow cytometry.

In certain embodiments of the methods disclosed herein, the isolatedplacental cells are one or more of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺,CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, MHC-I⁺ orABC-p⁺, where ABC-p is a placenta-specific ABC transporter protein (alsoknown as breast cancer resistance protein (BCRP) and as mitoxantroneresistance protein (MXR)). In another embodiment, the isolated placentalcells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, and OCT-4⁺. In another embodiment, theisolated placental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺,SH2⁺, SH3⁺, and SH4⁺. In another embodiment, the isolated placentalcells are CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺, SH3⁺, SH4⁺ andOCT-4⁺. In another embodiment, the isolated placental cells are CD10⁺,CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, MHC-1⁺, SH2⁺, SH3⁺,SH4⁺. In another embodiment, the isolated placental cells are OCT-4⁺ andABC-p⁺. In another embodiment, the isolated placental cells are SH2⁺,SH3⁺, SH4⁺ and OCT-4⁺. In another embodiment, the isolated placentalcells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻. In a specific embodiment,said OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻ cells are additionally CD10⁺,CD29⁺, CD34⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, and SH4⁺. Inanother embodiment, the isolated placental cells are OCT-4⁺ and CD34⁻,and either SH3⁺ or SH4⁺. In another embodiment, the isolated placentalcells are CD34⁻ and either CD10⁺, CD29⁺, CD44⁺, CD54⁺, CD90⁺, or OCT-4⁺.In certain embodiments, the isolated placental cells are CD10⁺, CD34⁻,CD105⁺ and CD200⁺.

In another embodiment, the isolated placental cells useful in themethods described herein are one or more of CD10⁺, CD29⁻, CD44⁺, CD45⁻,CD54/ICAM⁻, CD62-E⁻, CD62-L⁻, CD62-P⁻, CD80⁻, CD86⁻, CD103⁻, CD104⁻,CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(dim),CD184/CXCR4⁻,β2-microglobulin^(dim), MHC-I^(dim), MHC-II⁻, HLA-G^(dim),and/or PDL1^(dim). In certain embodiments, such placental cells are atleast CD29⁻ and CD54⁻. In another embodiment, such isolated placentalcells are at least CD44⁺ and CD106⁺. In another embodiment, suchisolated placental cells are at least CD29⁺.

In certain embodiments of any of the above characteristics, expressionof the cellular marker (e.g., cluster of differentiation or immunogenicmarker) is determined by flow cytometry. In certain other embodiments,expression of the cellular marker is determined by RT-PCR.

In another embodiment, said placental cells useful in the methodsdisclosed herein, e.g., said CD10⁺, CD34⁻, CD105⁺, CD200⁺ cells, expressone or more genes at a detectably higher level than an equivalent numberof bone marrow-derived mesenchymal stem cells, wherein said one or moregenes are one or more of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE,C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1,FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18,KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3,PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN,and ZC3H12A, and wherein said bone marrow-derived mesenchymal stem cellshave undergone a number of passages in culture equivalent to the numberof passages said isolated placental cells have undergone. In certainembodiments, said expression of said one ore more genes is determined,e.g., by RT-PCR or microarray analysis, e.g, using a U133-A microarray(Affymetrix). In another embodiment, said isolated placental cellsexpress said one or more genes when cultured for, e.g., anywhere fromabout 3 to about 35 population doublings, in a medium comprising 60%DMEM-LG (e.g., from Gibco) and 40% MCDB-201 (e.g., from Sigma); 2% fetalcalf serum (e.g., from Hyclone Labs.); 1× insulin-transferrin-selenium(ITS); 1× linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹ Mdexamethasone (e.g., from Sigma); 10⁻⁴ M ascorbic acid 2-phosphate(e.g., from Sigma); epidermal growth factor 10 ng/mL (e.g., from R&DSystems); and platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g.,from R&D Systems). In another embodiment, said isolated placental cellsexpress said one or more genes when cultured for from about 3 to about35 population doublings in a medium comprising 60% DMEM-LG (e.g., fromGibco) and 40% MCDB-201 (e.g., from Sigma); 2% fetal calf serum (e.g.,from Hyclone Labs.); 1× insulin-transferrin-selenium (ITS); 1× linoleicacid-bovine serum albumin (LA-BSA); 10⁻⁹ M dexamethasone (e.g., fromSigma); 10⁻⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (e.g., from R&D Systems); and platelet-derived growthfactor (PDGF-BB) 10 ng/mL (e.g., from R&D Systems).

In some embodiments, the placental cells express CD200 and do notexpress HLA-G, or express CD73, CD105, and CD200, or express CD200 andOCT-4, or express CD73 and CD105 and do not express HLA-G, or expressCD73 and CD105 and facilitate the formation of one or more embryoid-likebodies in a population of placental cells comprising said stem cell whensaid population is cultured under conditions that allow for theformation of an embryoid-like body, or express OCT-4 and facilitate theformation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow for the formation of anembryoid-like body.

In certain embodiments, the placental cells express CD200 and ARTS 1(aminopeptidase regulator of type 1 tumor necrosis factor); ARTS-1 andLRAP (leukocyte-derived arginine aminopeptidase); IL6 (interleukin-6)and TGFB2 (transforming growth factor, beta 2); IL6 and KRT18 (keratin18); IER3 (immediate early response 3), MEST (mesoderm specifictranscript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200 andKRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3 (nuclear factor(erythroid-derived 2)-like 3); or CD200 and TGFB2 at a detectably higherlevel than an equivalent number of bone marrow-derived mesenchymal stemcells (BM-MSCs) wherein said bone marrow-derived mesenchymal stem cellshave undergone a number of passages in culture equivalent to the numberof passages said isolated placental cells have undergone. In otherembodiments, the placental cells express ARTS-1, CD200, IL6 and LRAP;ARTS-1, IL6, TGFB2, IER3, KRT18 and MEST; CD200, IER3, IL6, KRT18, LRAP,MEST, NFE2L3, and TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP, MEST,NFE2L3, and TGFB2; or IER3, MEST and TGFB2 at a detectably higher levelthan an equivalent number of bone marrow-derived mesenchymal stem cellsBM-MSCs, wherein said bone marrow-derived mesenchymal stem cells haveundergone a number of passages in culture equivalent to the number ofpassages said isolated placental cells have undergone.

In various embodiments, said isolated placental cells useful in themethods disclosed herein, e.g., placental stem cells or placentalmultipotent cells, are contained within a population of cells, at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98% or 99% of the cells of which are said isolatedplacental cells. In certain other embodiments, the placental cells insaid population of cells are substantially free of cells having amaternal genotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% of the placental cells in saidpopulation have a fetal genotype, i.e., are fetal in origin. In certainother embodiments, the population of cells comprising said placentalcells are substantially free of cells having a maternal genotype; e.g.,at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99% of the cells in said population have a fetal genotype, i.e., arefetal in origin. In certain other embodiments, the population of cellscomprising said placental cells comprise cells having a maternalgenotype; e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells insaid population have a maternal genotype, i.e., are maternal in origin.

In an embodiment of any of the embodiments of placental cells (e.g.,PDACs) herein, the placental cells facilitate the formation of one ormore embryoid-like bodies in a population of placental cells comprisingsaid isolated placental cells when said population is cultured underconditions that allow the formation of an embryoid-like body e.g.,culture under proliferation conditions).

In certain embodiments of any of the placental cells disclosed herein,the cells are human.

In certain embodiments, any of the placental cells, e.g., placental stemcells or placental multipotent cells described herein, are autologous toa recipient. In certain other embodiments, any of the placental cells,e.g., placental stem cells or placental multipotent cells describedherein, are heterologous to a recipient.

In certain embodiments, the placental cells are cryopreserved prior tosaid administering. In another embodiment, said isolated placental cellsare obtained from a placental cell bank (e.g., a PDAC bank).

In any of the above embodiments of isolated placental cells, theisolated placental cells generally do not differentiate during culturingin growth medium, i.e., medium formulated to promote proliferation,e.g., during proliferation in growth medium. In another embodiment, saidisolated placental cells do not require a feeder layer in order toproliferate. In another embodiment, said isolated placental cells do notdifferentiate in culture solely as the result of culture in the absenceof a feeder cell layer.

In certain embodiments, said isolated placental cells are obtained byperfusion of a post-partum placenta that has been drained of blood andperfused to remove residual blood; drained of blood but not perfused toremove residual blood; or neither drained of blood nor perfused toremove residual blood. In another specific embodiment, said isolatedplacental cells are obtained by physical and/or enzymatic disruption ofplacental tissue.

Cell surface, molecular and genetic markers of placental cells, usefulin the methods provided herein, are described in detail in Section 4.2,below.

In specific embodiments, said individual to whom placental stem cellsare administered is additionally administered one or more of a secondtherapy (e.g., second therapeutic agent), wherein said second therapy(e.g., second therapeutic agent) comprises anti-inflammatory agents,steroids, immune suppressants, antibiotics, TNF-α inhibitors (e.g.,infliximab, adalimumab), PDE-4 inhibitors (e.g., apremiliast), IL-12inhibitors, IL-23 inhibitors, aviptadil, IFN-γ inhibitors, orantimalarials (e.g., chloroquine or hydroxychloroquine).

Examples of anti-inflammatory drugs useful in the treatment ofsarcoidosis, or sarcoidosis-related diseases, disorders or conditionsinclude, but are not limited to, mesalamine, 5-ASA (5-aminosalicylicacid) agents (e.g., ASACOL® (mesalamine, delayed-release), DIPENTUM(Osalazine), PENTASA® (mesalamine controlled-release)), sulfasalazine (acombination of 5-ASA and sulfapyridine), anti-inflammatory antibodies(e.g., Infliximab (REMICADE®)), and the like. Examples of steroidsuseful in the treatment of sarcoidosis, or sarcoidosis-related diseases,disorders or conditions include, but are not limited to, cortisone,hydrocortisone, predisone, methylprednisone, and the like. Typically, aspracticed in the art, the dosage of steroid is first delivered in arelatively large dose, followed by smaller dosages as inflammationsubsides. Examples of immune suppressants useful in the treatment ofsarcoidosis, or sarcoidosis-related diseases or disorders include, butare not limited to, cyclosporine A, 6-mercaptopurine or azathioprine.Any antibiotic can be used in the treatment of Crohn's disease,including, e.g., ampicillin, sulfonamide, cephalosporin, tetracycline,and/or metronidazole. In another specific embodiment, the second therapyis an administration of porcine whipworms, e.g., ova of Trichuris suis.

In another embodiment, administration of placental cells (e.g., PDACs),or therapeutic compositions comprising such cells, to an individual inneed thereof, can be accomplished, for local or systemic administration,by transplantation, implantation (e.g., of the cells themselves or thecells as part of a matrix-cell combination), injection (e.g., directlyto the site of the disease or condition, for example, directly to angranuloma), infusion, delivery via catheter, or any other means known inthe art for providing cell therapy. In other embodiment, placental cells(e.g., PDACs), or therapeutic compositions comprising such cells can bedelivered to an individual by intravenous, intraarterial,intraperitoneal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intrasynovial, intraocular, intravitreal(e.g., where there is an ocular involvement), intracerebral,intracerebroventricular (e.g., where there is a neurologic or braininvolvement), intracardiac (e.g., where there is a cardiac involvement),intrathecal, intraosseous infusion, intravesical, and transdermal,intracisternal, epidural, subcutaneous administration.

In specific embodiments, the placental cells are administered locally,e.g., directly into, or adjacent to (e.g., within 1-5 cm of) one or moredamaged tissues caused by, or relating to, sarcoidosis. In specificembodiments, the placental cells are administered intralesionally, e.g.,directly into, or adjacent to (e.g., within 1-5 cm of) one or morelesions or granulomas caused by, or relating to, sarcoidosis. In certainembodiments, the placental cells are administered in combination with amatrix, e.g., an injectable matrix. In certain other embodiments,placental cells are administered to an individual having asarcoidosis-related disease or disorder in combination with a solidmatrix, e.g., a bone substitute, a matrix or bone substitute describedin Section 4.7.4, below.

In another specific embodiment of the methods described above, saidisolated placental cells are administered by bolus injection. In anotherspecific embodiment, said isolated placental cells are administered byintravenous infusion. In a specific embodiment, said intravenousinfusion is intravenous infusion over about 1 to about 8 hours. Inanother specific embodiment, said isolated placental cells areadministered intracranially. In another specific embodiment, saidisolated placental cells are administered intraperitoneally. In anotherspecific embodiment, said isolated placental cells are administeredintra-arterially. In another specific embodiment of the method oftreatment, said isolated placental cells are administeredintramuscularly, intradermally, subcutaneously, or intraocularly.

In another embodiment of the methods described above, said isolatedplacental cells are administered by surgical implantation into saidindividual of a composition of matter comprising said isolated placentalcells. In a specific embodiment, said composition of matter is a matrixor scaffold. In another specific embodiment, said matrix or scaffold isa hydrogel. In another specific embodiment, said matrix or scaffold is adecellularized tissue. In another specific embodiment, said matrix orscaffold is a synthetic biodegradable composition. In another specificembodiment, said matrix or scaffold is a foam.

In another specific embodiment of the methods described above, saidisolated placental cells are administered once to said individual. Inanother specific embodiment, said isolated placental cells areadministered to said individual in two or more separate administrations.In another specific embodiment, said administering comprisesadministering between about 1×10⁴ and 1×10⁵ isolated placental cells,e.g., placental cells per kilogram of said individual. In anotherspecific embodiment, said administering comprises administering betweenabout 1×10⁵ and 1×10⁶ isolated placental cells per kilogram of saidindividual. In another specific embodiment, said administering comprisesadministering between about 1×10⁶ and 1×10⁷ isolated placental cells perkilogram of said individual. In another specific embodiment, saidadministering comprises administering between about 1×10⁷ and 1×10⁸isolated placental cells per kilogram of said individual. In otherspecific embodiments, said administering comprises administering betweenabout 1×10⁶ and about 2×10⁶ isolated placental cells per kilogram ofsaid individual; between about 2×10⁶ and about 3×10⁶ isolated placentalcells per kilogram of said individual; between about 3×10⁶ and about4×10⁶ isolated placental cells per kilogram of said individual; betweenabout 4×10⁶ and about 5×10⁶ isolated placental cells per kilogram ofsaid individual; between about 5×10⁶ and about 6×10⁶ isolated placentalcells per kilogram of said individual; between about 6×10⁶ and about7×10⁶ isolated placental cells per kilogram of said individual; betweenabout 7×10⁶ and about 8×10⁶ isolated placental cells per kilogram ofsaid individual; between about 8×10⁶ and about 9×10⁶ isolated placentalcells per kilogram of said individual; or between about 9×10⁶ and about1×10⁷ isolated placental cells per kilogram of said individual. Inanother specific embodiment, said administering comprises administeringbetween about 1×10⁷ and about 2×10⁷ isolated placental cells perkilogram of said individual to said individual. In another specificembodiment, said administering comprises administering between about1.3×10⁷ and about 1.5×10⁷ isolated placental cells per kilogram of saidindividual to said individual. In another specific embodiment, saidadministering comprises administering up to about 3×10⁷ isolatedplacental cells per kilogram of said individual to said individual. In aspecific embodiment, said administering comprises administering betweenabout 5×10⁶ and about 2×10⁷ isolated placental cells to said individual.In another specific embodiment, said administering comprisesadministering about 150×10⁶ isolated placental cells in about 20milliliters of solution to said individual.

In a specific embodiment, said administering comprises administeringbetween about 5×10⁶ and about 2×10⁷ isolated placental cells to saidindividual, wherein said cells are contained in a solution comprising10% dextran, e.g., dextran-40, 5% human serum albumin, and optionally animmunosuppressant.

In another specific embodiment, said administering comprisesadministering between about 5×10⁷ and 3×10⁹ isolated placental cellsintravenously. In specific embodiments, said administering comprisesadministering about 9×10⁸ isolated placental cells or about 1.8×10⁹isolated placental cells intravenously. In another specific embodiment,said administering comprises administering between about 5×10⁷ and 1×10⁸isolated placental cells intralesionally. In another specificembodiment, said administering comprises administering about 9×10⁷isolated placental cells intralesionally.

3.1 Definitions

As used herein, the term “about,” when referring to a stated numericvalue, indicates a value within plus or minus 10% of the stated numericvalue.

As used herein, the term “SH2” refers to an antibody that binds anepitope on the cellular marker CD105. Thus, cells that are referred toas SH2⁺ are CD105⁺.

As used herein, the terms “SH3” and SH4″ refer to antibodies that bindepitopes present on the cellular marker CD73. Thus, cells that arereferred to as SH3⁺ and/or SH4⁺ are CD73⁺.

A placenta has the genotype of the fetus that develops within it, but isalso in close physical contact with maternal tissues during gestation.As such, as used herein, the term “fetal genotype” means the genotype ofthe fetus, e.g., the genotype of the fetus associated with the placentafrom which particular isolated placental cells, as described herein, areobtained, as opposed to the genotype of the mother that carried thefetus. As used herein, the term “maternal genotype” means the genotypeof the mother that carried the fetus, e.g., the fetus associated withthe placenta from which particular isolated placental cells, asdescribed herein, are obtained.

As used herein, the term “granuloma” refers to a ball-like collection ofimmune cells formed as a result of inflammatory reaction, for instance,when the immune system attempts to eliminate substances (e.g.,infectious organisms such as bacteria and fungi as well as othermaterials such as keratin, suture fragments and vegetable particles)that it perceives as foreign but is unable to eliminate.

As used herein, the term “isolated cell,” e.g., “isolated placentalcell,” “isolated placental stem cell,” and the like, means a cell thatis substantially separated from other, different cells of the tissue,e.g., placenta, from which the stem cell is derived. A cell is“isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% ofthe cells, e.g., non-stem cells, with which the stem cell is naturallyassociated, or stem cells displaying a different marker profile, areremoved from the stem cell, e.g., during collection and/or culture ofthe stem cell.

As used herein, “multipotent,” when referring to a cell, means that thecell has the ability to differentiate into some, but not necessarilyall, types of cells of the body, or into cells having characteristics ofsome, but not all, types of cells of the body, or into cells of one ormore of the three germ layers. In certain embodiments, for example, anisolated placental cell (PDAC), as described in Section 4.2, below, thathas the capacity to differentiate into a cell having characteristics ofneurogenic, chondrogenic and/or osteogenic cells is a multipotent cell.

As used herein, the term “population of isolated cells” means apopulation of cells that is substantially separated from other cells ofa tissue, e.g., placenta, from which the population of cells is derived.

As used herein, the term “placental cell” refers to a stem cell orprogenitor cell that is isolated from a mammalian placenta, e.g., asdescribed in Section 4.2, below, or cultured from cells isolated from amammalian placenta, also referred to herein as “PDACs,” either as aprimary isolated or a cultured cell, regardless of the number ofpassages after a primary culture. In certain embodiments, the term“placental cells” as used herein does not, however, refer to, and theplacental cells used in the methods provided herein are not, however,trophoblasts, cytotrophoblasts, syncitiotrophoblasts, angioblasts,hemangioblasts, embryonic germ cells, embryonic stem cells, cellsobtained from an inner cell mass of a blastocyst, or cells obtained froma gonadal ridge of a late embryo, e.g., an embryonic germ cell. A cellis considered a “stem cell” if the cell displays attributes of a stemcell, e.g., a marker or gene expression profile associated with one ormore types of stem cells; the ability to replicate at least 10-40 timesin culture, and the ability to differentiate into cells displayingcharacteristics of differentiated cells of one or more of the three germlayers. Unless otherwise noted herein, the term “placental” includes theumbilical cord. The isolated placental cells disclosed herein, incertain embodiments, differentiate in vitro under differentiatingconditions, differentiate in vivo, or both.

As used herein, a placental cell is “positive” for a particular markerwhen that marker is detectable above background. Detection of aparticular marker can, for example, be accomplished either by use ofantibodies, or by oligonucleotide probes or primers based on thesequence of the gene or mRNA encoding the marker. For example, aplacental cell is positive for, e.g., CD73 because CD73 is detectable onplacental cells in an amount detectably greater than background (incomparison to, e.g., an isotype control). A cell is also positive for amarker when that marker can be used to distinguish the cell from atleast one other cell type, or can be used to select or isolate the cellwhen present or expressed by the cell. In the context of, e.g.,antibody-mediated detection, “positive,” as an indication a particularcell surface marker is present, means that the marker is detectableusing an antibody, e.g., a fluorescently-labeled antibody, specific forthat marker; “positive” also refers to a cell exhibiting the marker inan amount that produces a signal, e.g., in a cytometer, that isdetectably above background. For example, a cell is “CD200⁺” where thecell is detectably labeled with an antibody specific to CD200, and thesignal from the antibody is detectably higher than that of a control(e.g., background or an isotype control). Conversely, “negative” in thesame context means that the cell surface marker is not detectable usingan antibody specific for that marker compared a control (e.g.,background or an isotype control). For example, a cell is “CD34⁻” wherethe cell is not reproducibly detectably labeled with an antibodyspecific to CD34 to a greater degree than a control (e.g., background oran isotype control). Markers not detected, or not detectable, usingantibodies are determined to be positive or negative in a similarmanner, using an appropriate control. For example, a cell or populationof cells can be determined to be OCT-4⁺ if the amount of OCT-4 RNAdetected in RNA from the cell or population of cells is detectablygreater than background as determined, e.g., by a method of detectingRNA such as RT-PCR, slot blots, etc. Unless otherwise noted herein,cluster of differentiation (“CD”) markers are detected using antibodies.In certain embodiments, OCT-4 is determined to be present, and a cell is“OCT-4⁺” if OCT-4 is detectable using RT-PCR.

As used herein, the designation “low,” when referring to the expressionof a marker detectable in flow cytometry, means that the marker isexpressed by fewer than 10% of cells tested, or that fluorescenceattributable to the marker in, e.g., flow cytometry, is less than 1 logabove background.

As used herein, “treat” encompasses the remediation of, improvement of,lessening of the severity of, or reduction in the time course of, adisease, disorder or condition (e.g., sarcpodpsos), or any parameter orsymptom thereof.

4. DETAILED DESCRIPTION 4.1 Treatment of Sarcoidosis or Diseases,Disorders or Conditions Caused by, or Relating to, Sarcoidosis

Provided herein are methods of treating sarcoidosis or diseases ordisorders caused by, or relating to, sarcoidosis comprisingadministering an effective amount of tissue culture plastic-adherentplacental stem cells, e.g., PDACs, as described in Section 4.2, below.Methods for the treatment of such individuals, and for theadministration of such stem cells, alone or in combination with othertherapies, are discussed in detail below.

4.1.1 Sarcoidosis

Sarcoidosis is a multisystem granulomatous inflammatory disease. Thedisease is typically characterized by the formation of small, granularinflammatory lesions or granulomas (e.g., non-caseating granulomas) in avariety of organs, and/or the presence of immune responses (e.g.,presence of CD4⁺ T lymphocytes and macrophages) in affected tissues ororgans. Granulomatous inflammation may be attributed to the accumulationof monocytes, macrophages, a pronounced Th1 response and activatedT-lymphocytes, with elevated production of TNF-α, IL-2, IL-12, IFN-γ,IL-1, IL-6 or IL-15.

Sarcoidosis can be systemic (e.g., systemic sarcoidosis) or local (e.g.,localized sarcoid-like reactions). Granulomas can appear in almost anyorgan, and most often appear in the lungs or the lymph nodes. Othercommon sites include the liver, spleen, skin and eyes. The involvementof a specific organ may be mild or severe, self-limited or chronic, andlimited or wide-ranging in extent. Symptoms usually appear gradually butcan occasionally appear suddenly. The common symptoms experienced bysarcoidosis patients comprise fatigue, shortness of breath, cough thatwill not go away, skin lesions or skin rashes on face, arms, or shins,inflammation of the eyes, weight loss, night sweats, dyspnea, cough,chest discomfort, crackles. malaise, weakness, anorexia, weight loss, orfever. Other symptoms include, for example, enlarged lymph glands(armpit lump), enlarged liver, enlarged spleen, dry mouth or nosebleed.Symptoms of different types of sarcoidosis are described below.

The clinical course generally varies. In some embodiments, sarcoidosiscan be asymptomatic. In other embodiments, a tissue or organ canremained inflamed or become scarred or fibrotic if one or moregranulomas in the tissue or organ do not heal. In still otherembodiments, sarcoidosis can result in a debilitating chronic conditionthat may lead to death (e.g., irreversible pulmonary fibrosis).

Thus, in one aspect, the PDACs, populations of such cells, andpopulations of cells comprising PDACs, provided herein can be used totreat individuals exhibiting sarcoidosis, or one or more diseases,disorders or conditions caused by, or relating to, sarcoidosis. In aspecific embodiment, provided herein is a method of treating anindividual having sarcoidosis, comprising administering to theindividual isolated placental cells, a population of isolated placentalcells, or a population of cells comprising isolated placental cells,wherein said administration results in the detectable reduction ofprogression, detectable lessening of worsening, and/or detectableimprovement, of one or more symptoms of said sarcoidosis. In a specificembodiment, said therapeutically effective amount of placental cellsreduces, e.g., detectably reduces, the number of, or degree of severityof, or reduces the rate of increase in the number of, or degree ofseverity, one or more granulomas in said individual. In another specificembodiment, said therapeutically effective amount of placental cellsreduces, e.g., detectably reduces, the number of, or reduces the rate ofincrease in the number of, monocytes, macrophages, or activatedT-lymphocytes. In another specific embodiment, said therapeuticallyeffective amount of placental cells reduces, e.g., detectably reduces,the level of, or reduces the rate of increase in the level of, one ormore TNF-α, IL-2, IL-12, IFN-γ, IL-1, IL-6, IL-15, or Th1 response insaid individual.

In certain embodiments, said sarcoidosis is one or more of systemicsarcoidosis, cutaneous sarcoidosis, Löfgren's syndrome,neurosarcoidosis, pulmonary sarcoidosis, cardiac sarcoidosis, ocularsarcoidosis, hepatic sarcoidosis, musculoskeletal sarcoidosis, renalsarcoidosis, or sarcoidosis with the involvement of other organs ortissues.

Systemic sarcoidosis—is sarcoidosis with multiple organ involvement. Inone embodiment, provided herein is a method of treating an individualhaving systemic sarcoidosis, comprising administering to the individualisolated placental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said systemic sarcoidosis. In some embodiments, saidone or more symptoms of systemic sarcoidosis comprise non-specificgeneral symptoms such as weight loss, fatigue, loss of appetite, fever,chills, night sweats, formation of granulomas, fatigue, aches, pains orarthritis.

In other embodiments, systemic sarcoidosis can present with specificsymptoms related to a particular organ affected (e.g., dry eyes,swelling of the knees, blurry vision, shortness of breath, cough, skinlesions such as rashes etc.). In specific embodiments, said specificsymptom is one or more of a pulmonary, pulmonary lymphatic,musculoskeletal, hepatic, joint, hematologic, dermatologic, ocular,psychiatric, neurological, renal, splenic, neurologic, nasal sinus,cardiac, bone, oral, gastric, intestinal, endocrine, pleural orreproductive symptom.

Cutaneous sarcoidosis—is a complication of sarcoidosis with skininvolvement. In another embodiment, provided herein is a method oftreating an individual having cutaneous sarcoidosis, comprisingadministering to the individual isolated placental cells, a populationof isolated placental cells, or a population of cells comprisingisolated placental cells, wherein said administration results in thedetectable reduction of progression, detectable lessening of worsening,and/or detectable improvement, of one or more symptoms of said cutaneoussarcoidosis. In certain embodiments, the cutaneous sarcoidosis comprisesannular sarcoidosis, erythrodermic sarcoidosis, ichthyosiformsarcoidosis, hypopigmented sarcoidosis, morpheaform sarcoidosis, mucosalsarcoidosis, papular sarcoid, scar sarcoid, subcutaneous sarcoidosis andulcerative sarcoidosis. In some embodiments, the one or more symptoms ofcutaneous sarcoidosis comprise a variety of skin lesions or conditions,either specific or non-specific (e.g., similar to several other skinconditions). Exemplary skin lesions or conditions associated withcutaneous sarcoidosis comprise papules (e.g., granulomatous rosacea,acne or benign appendageal tumors), skin plaques (e.g., psoriasis,lichen planus, nummular eczema, discoid lupus erythematosus, granulomaannulare, cutaneous T-cell lymphoma, Kaposi's sarcoma or secondarysyphilis), lupus pernio (e.g., scar or discoid lupus erythematosus),erythema nodosum (e.g., raised, red, firm skin sores, cellulitis,furunculosis or other inflammatory panniculitis), maculopapulareruptions, nodular lesions deeper in the skin or infiltration of oldscars. Other skin symptoms include, for example, skin rashes, old scarsbecome more raised, skin lesions or hair loss.

Löfgren's syndrome—represents an acute presentation of systemicsarcoidosis, which is typically characterized by the triad of erythemanodosum, bilateral hilar denopathy and arthritis or arthralgias. It mayalso be accompanied by fever. In another embodiment, provided herein isa method of treating an individual having Löfgren's syndrome, comprisingadministering to the individual isolated placental cells, a populationof isolated placental cells, or a population of cells comprisingisolated placental cells, wherein said administration results in thedetectable reduction of progression, detectable lessening of worsening,and/or detectable improvement, of one or more symptoms of said Löfgren'ssyndrome. In specific embodiments, the one or more symptoms compriseerythema nodosum, bilateral hilar denopathy, arthritis, arthralgias orfever.

Neurosarcoidosis or neurosarcoid—refers to sarcoidosis in whichinflammation and abnormal deposits occur in the brain, spinal cord, andany other areas of the nervous system. In another embodiment, providedherein is a method of treating an individual having neurosarcoidosis,comprising administering to the individual isolated placental cells, apopulation of isolated placental cells, or a population of cellscomprising isolated placental cells, wherein said administration resultsin the detectable reduction of progression, detectable lessening ofworsening, and/or detectable improvement, of one or more symptoms ofsaid neurosarcoidosis. Neurosarcoidosis may affect any part of thenervous system, for instance, nerves to the muscles of the face (cranialnerve VII), which may lead to symptoms of facial weakness (e.g., facialpalsy), nerves in the eye or nerves that control taste, smell, orhearing.

In some embodiments, the symptoms of neurosarcoidosis comprise changesin menstrual periods, excessive tiredness (e.g., fatigue), headache,visual changes, retinopathy, radicular pain, loss of bowel or bladdercontrol, carpal tunnel syndrome, and/or paraplegia, excessive thirst orhigh urine output. In other embodiments, the symptoms ofneurosarcoidosis comprise confusion, disorientation, decreased hearing,dementia or delirium, dizziness or vertigo (e.g., abnormal sensation ofmovement), double vision or other vision problems, facial palsy(weakness, drooping), headache, loss of sense of smell or taste,abnormal tastes, psychiatric disturbances, seizures or speechimpairment, muscle weakness or sensory losses, or in some occasions,hypopituiarism. In specific embodiments, the symptoms ofneurosarcoidosis comprise formation of granulomas in the nervous system(e.g., brain, spinal cord, or facial and optic nerves), headache,confusion, malaise or facial paralysis.

Pulmonary sarcoidosis—refers to sarcoidosis that affects pulmonarytissues or organs (e.g., lungs). In another embodiment, provided hereinis a method of treating an individual having pulmonary sarcoidosis,comprising administering to the individual isolated placental cells, apopulation of isolated placental cells, or a population of cellscomprising isolated placental cells, wherein said administration resultsin the detectable reduction of progression, detectable lessening ofworsening, and/or detectable improvement, of one or more symptoms ofsaid pulmonary sarcoidosis. The symptoms of pulmonary sarcoidosisusually involve lung and/or chest symptoms, which can be determined by,for examples, lung gallium (Ga.) scan, chest X-ray, pulmonary functiontests, exercise pulse oximetry, CT scan of chest, PET scan, CT-guidedbiopsy, mediastinoscopy, open lung biopsy or bronchoscopy with biopsy.

In certain embodiments, the symptoms of pulmonary sarcoidosis comprisegranulomas in alveolar septa, bronchiolar, and bronchial walls,shortness of breath, cough, loss of lung volume and abnormal lungstiffness, abnormal or deteriorating lung function, decrease in lungvolume, decreased compliance, scarring of lung tissue, or bleeding fromthe lung tissue. Other symptoms include, for example, limited amount ofair drawn into the lungs, higher than normal expiratory flow ratios,decreased vital capacity (full breath in, to full breath out), increasedFEV₁/FVC ratio, obstructive lung changes, which can cause a decrease inthe amount of air that can be exhaled, or enlarged lymph nodes in thechest, which can compress airways or when internal inflammation ornodules impede airflow, pulmonary hypertension, or pulmonary failure.

Without being bound by any theory, the Scadding criteria are themeasures most commonly used for disease staging of patients who havepulmonary sarcoidosis. Briefly, the radiographic evidence for each stagecan be described as follows: Stage I: bilateral hilar and/or mediastinallymphadenopathy (enlarged lymph nodes); Stage II: bilateral hilar and/ormediastinal lymphadenopathy; evidence of pulmonary infiltrates; StageIII: alterations in the parenchymal tissue; no lymph node enlargement;and Stage IV: Evidence of pulmonary fibrosis. Thus, provided herein is amethod of treating an individual having pulmonary sarcoidosis ordisease, disorder or condition caused by, or relating to pulmonarysarcoidosis, comprising administering to the individual isolatedplacental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said pulmonary sarcoidosis as determined by theScadding criteria.

In some embodiments, pulmonary sarcoidosis can develop into pulmonaryfibrosis (e.g., irreversible pulmonary fibrosis), which can distort thestructure of the lungs and impair breathing or bronchiectasis, a lungdisease characterized by destruction and widening of the large airways.Thus, in a specific embodiment, said disease or disorder is pulmonaryfibrosis or bronchiectasis. In a more specific embodiment, providedherein is a method of treating an individual having pulmonary fibrosis(e.g., irreversible pulmonary fibrosis) caused by, or relating tosarcoidosis, comprising administering to the individual isolatedplacental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said pulmonary fibrosis (e.g., irreversiblepulmonary fibrosis). In another specific embodiment, provided herein isa method of treating an individual having bronchiectasis caused by, orrelating to sarcoidosis, comprising administering to the individualisolated placental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said bronchiectasis.

In other embodiments, sarcoidosis can involve pulmonary lymphatic systemsuch as hilar or mediastinal involvement, as determined by chest x-ray,and present with symptoms of nontender peripheral or cervicallymphadenopathy.

Cardiac sarcoidosis—refers to sarcoidosis with myocardial involvement.In another embodiment, provided herein is a method of treating anindividual having cardiac sarcoidosis, comprising administering to theindividual isolated placental cells, a population of isolated placentalcells, or a population of cells comprising isolated placental cells,wherein said administration results in the detectable reduction ofprogression, detectable lessening of worsening, and/or detectableimprovement, of one or more symptoms of said cardiac sarcoidosis.

In some embodiments, the one or more symptoms of cardiac sarcoidosis aresimilar to the symptoms of a disease or disorder of the circulatorysystem, for example, myocardial infarction, cardiomyopathy, aneurysm,angina, aortic stenosis, aortitis, arrhythmias, arteriosclerosis,arteritis, asymmetric septal hypertrophy (ASH), atherosclerosis, atrialfibrillation and flutter, bacterial endocarditis, Barlow's Syndrome(mitral valve prolapse), bradycardia, Buerger's Disease (thromboangiitisobliterans), cardiomegaly, carditis, carotid artery disease, coarctationof the aorta, congenital heart defects, congestive heart failure,coronary artery disease, Eisenmenger's Syndrome, embolism, endocarditis,erythromelalgia, fibrillation, fibromuscular dysplasia, heart block,heart murmur, hypertension, hypotension, idiopathic infantile arterialcalcification, Kawasaki Disease (mucocutaneous lymph node syndrome,mucocutaneous lymph node disease, infantile polyarteritis), metabolicsyndrome, microvascular angina, myocarditis, paroxysmal atrialtachycardia (PAT), periarteritis nodosa (polyarteritis, polyarteritisnodosa), pericarditis, peripheral vascular disease, critical limbischemia, phlebitis, pulmonary valve stenosis (pulmonic stenosis),Raynaud's Disease, renal artery stenosis, renovascular hypertension,rheumatic heart disease, diabetic vasculopathy, septal defects, silentischemia, syndrome X, tachycardia, Takayasu's Arteritis, Tetralogy ofFallot, transposition of the great vessels, tricuspid atresia, truncusarteriosus, valvular heart disease, varicose ulcers, varicose veins,vasculitis, ventricular septal defect, Wolff-Parkinson-White Syndrome,endocardial cushion defect, acute rheumatic fever, acute rheumaticpericarditis, acute rheumatic endocarditis, acute rheumatic myocarditis,chronic rheumatic heart diseases, diseases of the mitral valve, mitralstenosis, rheumatic mitral insufficiency, diseases of aortic valve,diseases of other endocardial structures, ischemic heart disease (acuteand subacute), angina pectoris, acute pulmonary heart disease, pulmonaryembolism, chronic pulmonary heart disease, kyphoscoliotic heart disease,myocarditis, endocarditis, endomyocardial fibrosis, endocardialfibroelastosis, atrioventricular block, cardiac dysrhythmias, myocardialdegeneration, cerebrovascular disease, a disease of arteries, arteriolesand capillaries, or a disease of veins and lymphatic vessels. Thus, incertain embodiments, improvement in an individual having sarcoidosis ora sarcoidosis-related disease or disorder, wherein the individual isadministered the PDACs or therapeutic compositions provided herein, canbe assessed or demonstrated by detectable improvement in one or moresymptoms of said sarcoidosis or said sarcoidosis-related disease ordisorder.

In certain embodiments, the method of comprises administering placentalcells to said individual in an amount and for a time sufficient fordetectable improvement of one or more indicia of cardiac function,wherein said indicia of cardiac function are chest cardiac output (CO),cardiac index (CI), pulmonary artery wedge pressure (PAWP), cardiacindex (CI), % fractional shortening (% FS), ejection fraction (EF), leftventricular ejection fraction (LVEF); left ventricular end diastolicdiameter (LVEDD), left ventricular end systolic diameter (LVESD),contractility (dP/dt), a decrease in atrial or ventricular functioning,an increase in pumping efficiency, a decrease in the rate of loss ofpumping efficiency, a decrease in loss of hemodynamic functioning, ordecrease in complications associated with cardiomyopathy, as compared tothe individual prior to administration of said PDACs.

In specific embodiments of the methods of treatment provided herein, thePDACs are administered with stem cells (that is, stem cells that are notPDACs), myoblasts, myocytes, cardiomyoblasts, cardiomyocytes, orprogenitors of myoblasts, myocytes, cardiomyoblasts, and/orcardiomyocytes.

In a specific embodiment, the methods of treatment provided hereincomprise administering PDACs, e.g., a therapeutic composition comprisingthe cells, to a patient with sarcoidosis or a disease, disorder orcondition caused by, or relating to, sarcoidosis; and evaluating thepatient for improvements in cardiac function, wherein the therapeuticcell composition is administered as a matrix-cell complex. In certainembodiments, the matrix is a scaffold, preferably bioabsorbable,comprising at least the cells.

To this end, further provided herein are populations of PDACs contactedwith, e.g., incubated or cultured in the presence of, one or morefactors that stimulate stem or progenitor cell differentiation along acardiogenic, angiogenic, hemangiogenic, or vasculogenic pathway. Suchfactors include, but are not limited to factors, such as growth factors,chemokines, cytokines, cellular products, demethylating agents, andother factors which are now known or later determined to stimulatedifferentiation, for example of stem cells, along cardiogenic,angiogenic, hemangiogenic, or vasculogenic pathways or lineages.

In certain embodiments, PDACs may be differentiated along cardiogenic,angiogenic, hemangiogenic, or vasculogenic pathways or lineages in vitroby culture of the cells in the presence of factors comprising at leastone of a demethylation agent, a BMP (bone morphogenetic protein), FGF(fibroblast growth factor), Wnt factor protein, Hedgehog protein, and/oran anti-Wnt factor.

PDACs, and populations of such cells, can be provided therapeutically orprophylactically to an individual, e.g., an individual having a disease,disorder or condition of, or affecting, the heart or circulatory systemrelating to sarcoidosis. Such diseases, disorders or conditions caninclude congestive heart failure due to atherosclerosis, cardiomyopathy,or cardiac injury, e.g., an ischemic injury, such as from myocardialinfarction or wound (acute or chronic).

Ocular Sarcoidosis—is sarcoidosis that affects the eye. In anotherembodiment, provided herein is a method of treating an individual havingocular sarcoidosis, comprising administering to the individual isolatedplacental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said ocular sarcoidosis. In certain embodiments, theone or more symptoms of ocular sarcoidosis comprise uveitis (e.g.,granulomatous uvetis), uveoparotitis, retinal inflammation, loss ofvisual acuity, blindness red, watery eyes, iris nodules,retinochoroiditis, conjunctivitis, lacrimal gland involvement orproptosis.

Sarcoidosis with Musculoskeletal, Hepatic, Hematologic, Psychiatric,Renal, Splenic, Nasal Sinus, Oral, Gastric or Intestinal, Endocrine,Pleural or Reproductive Involvement

In certain embodiments, sarcoidosis can involve muscle, hepatic, joint,hematologic, psychiatric, renal, splenic, nasal sinus, bone, oralgastric or intestinal, endocrine, pleural or reproductive system andpresent with respective symptoms. In another embodiment, provided hereinis a method of treating an individual having sarcoidosis withmusculoskeletal, hepatic, joint, hematologic, psychiatric, renal,splenic, nasal sinus, oral gastric or intestinal, endocrine, pleural orreproductive involvement, comprising administering to the individualisolated placental cells, a population of isolated placental cells, or apopulation of cells comprising isolated placental cells, wherein saidadministration results in the detectable reduction of progression,detectable lessening of worsening, and/or detectable improvement, of oneor more symptoms of said sarcoidosis.

In one embodiment, sarcoidosis with musculoskeletal involvement can beasymptomatic with or without enzyme elevations, or present with symptomsof, for example, insidious or acute myopathy with muscle weakness,arthritis (e.g., ankle, knee, wrist, and elbow arthritis), chronicarthritis with Jaccoud's deformities or dactylics, periarthritis,arthralgia, osteolytic or cystic lesions, osteopenia or Löfgren'ssyndrome. In another embodiment, sarcoidosis with hepatic involvementcan be aymptomatic, or present with one or more symptoms such as mildelevations in liver function test results, hypolucent lesions on CTscans with radiopaque dye, hepatomegaly, changes in the liver enzymelevels, liver diseases, fever, malaise, fatigue, cholestasis, cirrhosis,or with symptoms similar to granulomatous hepatitis.

In another embodiment, patients with hematologic sarcoidosis have one ormore symptoms of lymphopenia, anemia of chronic disease, anemia due togranulomatous infiltration of bone marrow, pancytopenia, splenicsequestration, thrombocytopenia or leucopenia. In another embodiment,patients with sarcoidosis can exhibit psychiatric symptoms (e.g.,depression). In another embodiment, sarcoidosis with renal involvementcan present with symptoms of asymptomatic hypercalciuria, interstitialnephritis, chronic renal failure caused by nephrolithiasis, ornephrocalcinosis. In another embodiment, sarcoidosis with splenicinvolvement can be asymptomatic, or present with symptoms of pain,thrombocytopenia or as determined by x-ray or CT scan. In anotherembodiment, sarcoidosis with nasal sinus involvement can present withsymptoms of sinus mucosa with symptoms similar to common allergic andinfectious sinusitis, or upus pernio. In another embodiment, sarcoidosiswith oral involvement can present with symptoms of asymptomatic parotidswelling, parotitis with xerostomia, Heerfordt's syndrome, uveitis,bilateral parotid swelling, facial palsy, chronic fever, oral lupuspernio, or disfigured hard palate, cheek, tongue, and gums. In anotherembodiment, symptoms of sarcoidosis with gastric or intestinal,endocrine, pleural or reproductive involvement comprise gastricgranulomas, mesenteric lymphadenopathy, abdominal pain,panhypopituitarism, thyroid infiltration, secondary hypoparathyroidism,hypercalcemia, or lymphocytic exudative effusions.

In another embodiment, provided herein is a method of treating anindividual having sarcoidosis with the involvement of one or moretissues or organs other than pulmonary tissues or organs, comprisingadministering to the individual isolated placental cells, a populationof isolated placental cells, or a population of cells comprisingisolated placental cells, wherein said administration results in thedetectable reduction of progression, detectable lessening of worsening,and/or detectable improvement, of one or more symptoms of saidsarcoidosis. In another embodiment, provided herein is a method oftreating an individual having sarcoidosis with the involvement of one ormore tissues or organs other than the lungs, comprising administering tothe individual isolated placental cells, a population of isolatedplacental cells, or a population of cells comprising isolated placentalcells, wherein said administration results in the detectable reductionof progression, detectable lessening of worsening, and/or detectableimprovement, of one or more symptoms of said sarcoidosis.

Other Diseases, Disorders or Conditions

In some embodiments, said sarcoidosis-related disease or disorder is acancer (e.g., a cancer in an organ known to be affected in sarcoidosis).In another embodiment, provided herein is a method of treating anindividual having sarcoidosis-related cancer, comprising administeringto the individual isolated placental cells, a population of isolatedplacental cells, or a population of cells comprising isolated placentalcells, wherein said administration results in the detectable reductionof progression, detectable lessening of worsening, and/or detectableimprovement, of one or more symptoms of said sarcoidosis-related cancer.

Without being bound by any theory, patients with sarcoidosis can have anincreased risk for cancers (e.g., lung cancer or malignant lymphomas).In certain embodiment, said cancer is a solid tumor. In someembodiments, said cancer is primary ductal carcinoma, leukemia, acute Tcell leukemia, chronic myeloid lymphoma (CML), acute myelogenousleukemia, chronic myelogenous leukemia (CML), lung carcinoma, colonadenocarcinoma, histiocytic lymphoma, colorectal carcinoma, colorectaladenocarcinoma, or retinoblastoma. In a specific embodiments, the canceris hairy cell leukemia, acute myeloid leukemia (acute myelogenousleukemia) or acute myeloblastic leukemia.

In other embodiments, said sarcoidosis-related disease or disordercomprises vitamin D dysregulation, hyperprolactinemia, thyroid diseases(e.g., hypothyroidism, hyperthyroidism and thyroid autoimmunity),autoimmune disorders or sarcoidosis-lymphoma syndrome andhypersensitivity (e.g., celiac disease, type IV hypersensitivity).

Typically, an individual presenting with one or more symptoms ofsarcoidosis is assessed for sarcoidosis at least once before a finaldiagnosis of sarcoidosis, e.g., as a part of tests performed to arriveat a diagnosis of sarcoidosis. Also, typically, an individual diagnosedwith sarcoidosis is assessed at least once, usually more than once,after a diagnosis of sarcoidosis, for symptoms of sarcoidosis to gaugeprogress of the disease. Such an assessment may comprise diagnosis ofpulmonary functions or symptoms, e.g., chest imaging, chest X-ray, CTscan of chest, PET scan, CT-guided biopsy, mediastinoscopy, open lungbiopsy, bronchoscopy with biopsy, endobronchial ultrasound or endoscopicultrasound with FNA of mediastinal lymph nodes; tissue biopsy, e.g.,tissue biopsy, angiotensin-converting enzyme blood; magnetic resonanceimaging (MRI), e.g., to detect cardiac, neurological, or liverinvolvement, computerized tomography (CT) scanning, positron emissiontomography (PET) scanning, electrocardiogram of affected tissues ororgans; liver function tests; ocular examination; blood, ionize andurine calcium determination; serum phosphorus determination; thyroidfunction tests; slit eye lamp exam to detec uveitis; or exclusion ofother granulomatous disorders. Effectiveness of treatment ofsarcoidosis, e.g., effectiveness of administering placental cells, canbe assessed by any one, or more, of such symptoms of sarcoidosis.Effectiveness can also be assessed by monitoring the reduction ofprogression, detectable lessening of worsening, and/or detectableimprovement of symptoms or functions of affected organs in saidindividual, before and after administration of said placental cells.

In one aspect, provided herein are methods for treating a patient withsarcoidosis or a sarcoidosis-related disease, disorder or conditioncomprising administering a therapeutic cell composition to a patientwith sarcoidosis or a sarcoidosis-related disease, disorder orcondition, and evaluating the patient for improvements in the symptomsof sarcoidosis, wherein said cell composition comprises PDACs asdescribed herein.

In one embodiment, said methods can comprise determining, once or aplurality of times before said administering, and/or once or a pluralityof times after said administering, one or more of (1) a number and/ordegree of affected organs in said individual; (2) a number and/or degreeof symptoms in said individual, (3) a number and/or degree ofgranulomas; or (4) the degree of inflammation in said individual.

In certain embodiments, said therapeutically effective amount ofplacental cells reduces, e.g., detectably reduces, the number of, ordegree of severity of, or reduces the rate of increase in the number of,or degree of severity, said one or more symptoms in said individuals,and/or results in detectable improvement, of one or more symptoms ofsarcoidosis or sarcoidosis-related diseases, disorders or conditions.

In one embodiment, said therapeutically effective amount is an amountthat results in a detectable improvement in at least one symptom of saidsarcoidosis or a sarcoidosis-related disease or disorder. In anotherembodiment, said therapeutically effective amount is an amount thatresults in modulation, e.g., suppression, of the activity, e.g.,proliferation, of an immune cell, or plurality of immune cells. In aspecific embodiment, said therapeutically effective amount is an amountthat results in suppression of an inflammatory response, as determinedby one or more inflammatory markers (e.g., C-reactive protein, IL-6, oras determined by PLAC test). In a specific embodiment, said modulation(e.g., suppression) is performed by contacting the immune cell(s) with aplurality of placental stem cells for a time sufficient for saidplacental stem cells to detectably modulate (e.g., suppress) an immuneresponse. In another embodiment, said therapeutically effective amountis an amount that, when transplanted to an affected tissue or organ insaid individual, allows the formation of colony-forming units in theaffected tissue or organ.

In some embodiments, said therapeutically effective amount of placentalcells results in, e.g., detectably results in, the lung functions, e.g.,as determined by chest imaging, chest X-ray, CT scan of chest, PET scanor CT-guided biopsy. In other embodiments, said therapeuticallyeffective amount of placental cells reduces the number and/or degree ofskin lesions, e.g., as determined by excisional biopsy. In still otherembodiments, when a cancer is involved, said placental cells reduce thenumber of cancerous cells, by at least, e.g., 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or99%, compared to the number of said cells prior to administration ofsaid placental cells.

In another embodiment, treatment comprises treatment of an individualwith sarcoidosis or a disease, disorder or condition caused by, orrelating to sarcoidosis, with a therapeutic cell composition comprisingplacental cells (e.g., PDACs), either with or without another cell type.In certain embodiments, the individual experiences benefits from thetherapy, for example from the ability of the cells to support the growthof other cells, including stem cells or progenitor cells present in theheart, from the tissue ingrowth or vascularization of the tissue, andfrom the presence of beneficial cellular factors, chemokines, cytokinesand the like, but the cells do not integrate or multiply in theindividual. In another embodiment, the patient benefits from thetherapeutic treatment with the cells, but the cells do not survive for aprolonged period in the patient. In one embodiment, the cells graduallydecline in number, viability or biochemical activity, in otherembodiments, the decline in cells may be preceded by a period ofactivity, for example growth, division, or biochemical activity. Inother embodiments, senescent, nonviable or even dead cells are able tohave a beneficial therapeutic effect.

Improvement in an individual having sarcoidosis or a disease, disorderor condition caused by, or relating to sarcoidosis, wherein theindividual is administered the PDACs or therapeutic compositionsprovided herein, can be assessed or demonstrated by detectableimprovement in one or more symptoms of said sarcoidosis or saidsarcoidosis-related disease or disorder.

Improvement in an individual receiving the PDACs, or therapeuticcompositions comprising PDACs, provided herein can also be assessed bysubjective metrics, e.g., the individual's self-assessment about his orher state of health following administration.

Success of administration of the cells is not, in certain embodiments,based on survival in the individual of the administered PDACs. Successis, instead, based on one or more metrics of improvement in one or moresymptoms of sarcoidosis, as noted above. Thus, the cells need notintegrate and beat with the patient's affected tissues or organs.

4.1.2 Administration of Placental Cells

In some embodiments, administration of placental cells (e.g., PDACs), ortherapeutic compositions comprising such cells, to an individual in needthereof, can be accomplished, for local or systemic administration, bytransplantation, implantation (e.g., of the cells themselves or thecells as part of a matrix-cell combination), injection (e.g., directlyto the site of the disease or condition, for example, directly to one ormore lesions or granulomas caused by, or relating to sarcoidosis),infusion, delivery via catheter, or any other means known in the art forproviding cell therapy. In other embodiment, placental cells (e.g.,PDACs), or therapeutic compositions comprising such cells can bedelivered to an individual by intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, intrasynovial, andsubcutaneous administration.

In one embodiment, the therapeutic cell compositions are provided to anindividual in need thereof, for example, by injection into one or moresites in the individual. In a specific embodiment, when there is cardiacinvolvement, the therapeutic cell compositions are provided byintracardiac injection, e.g., to an ischemic area in the heart. In otherspecific embodiments, the cells are injected onto the surface of theaffected organ, into an adjacent area, or even to a more remote area. Inpreferred embodiments, the cells can home to the diseased or injuredarea.

In specific embodiments, the placental cells are administeredintralesionally, e.g., directly into, or adjacent to (e.g., within 1-5cm of) one or more lesions or granulomas caused by, or relating tosarcoidosis. In certain embodiments, the placental cells areadministered in combination with a matrix, e.g., an injectable matrix.In certain other embodiments, placental cells are administered to anindividual having sarcoidosis or a sarcoidosis-related disease ordisorder in combination with a solid matrix, e.g., a bone substitute, amatrix or bone substitute described in Section 4.7.4, below.

In certain other embodiments, the placental cells are administeredintravenously to the individual. The placental cells can be administeredfrom any container, and by any delivery system, medically suitable forthe delivery of fluids, e.g., fluids comprising cells, to an individual.Such containers can be, for example, a sterile plastic bag, flask, jar,or other container from which the placental cell population can beeasily dispensed. For example, the container can be a blood bag or otherplastic, medically-acceptable bag suitable for the intravenousadministration of a liquid to a recipient.

Intralesional or intravenous administration can comprise, e.g., about,at least, or no more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or more isolatedplacental cells in a single dose. The isolated placental cells may beadministered once, or more than once, during a course of therapy.Preferably, the administered placental cells comprise 50% viable cellsor more (that is, at least about 50% of the cells in the population arefunctional or living). Preferably, at least about 60% of the cells inthe population are viable. More preferably, at least about 70%, 80%,90%, 95%, or 99% of the cells in the population in the pharmaceuticalcomposition are viable.

In certain embodiments, the individual is administered a therapeuticallyeffective amount of PDACs, e.g., in a population of cells that comprisethe PDACs. In a specific embodiment, the population comprises about 50%PDACs. In another specific embodiment, the population is a substantiallyhomogeneous population of PDACs. In other embodiments the populationcomprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 95%, 98% or 99% PDACs.

An individual having sarcoidosis or a sarcoidosis-related disease orcondition can be administered PDACs at any time the cells would betherapeutically beneficial. In certain embodiments, for example, thePDACs or therapeutic compositions of the invention are administeredwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 days of the myocardial infarction. Administration proximal in timeto a myocardial infarction, e.g., within 1-3 or 1-7 days, is preferableto administration distal in time, e.g., after 3 or 7 days after amyocardial infarction. In other embodiments, the cells or therapeuticcompositions of the invention are administered within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days of initialdiagnosis of the disease or condition.

In certain embodiments, the individual to be treated is a mammal. In aspecific embodiment the individual to be treated is a human of any ageand/or at any clinical stage or sarcoidosis or a sarcoidosis-relateddisease, disorder or condition. In specific embodiments, the individualis a livestock animal or a domestic animal. In other specificembodiments, the individual to be treated is a horse, sheep, cow orsteer, pig, dog or cat.

4.1.3 Combination Therapies

Treatment of sarcoidosis or a sarcoidosis-related disease or disorder,e.g., systemic sarcoidosis, can comprise administration of isolatedplacental cells, in particular, the isolated placental cells describedin detail in Section 4.2, below (PDACs), in combination with one or moreof a second therapy (e.g., second therapeutic agent), to the individualhaving sarcoidosis or a sarcoidosis-related disease or disorder.

The combination therapies provided herein do not restrict the order inwhich therapies are administered to a subject. For example, a firsttherapy can be administered before (e.g., 1 minute, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g., 1minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) theadministration of a second therapy to a subject which had, has, or issusceptible to a given disease. Any additional therapy can beadministered in any order with the other additional therapies.

In certain embodiments, the second therapeutic agent comprises TNF-αinhibitors (e.g., infliximab or adalimumab), antimalarials (e.g.,chloroquine or hydroxychloroquine), thalidomide, azathioprine,mycophenolate, cyclophosphamide, chlorambucil, methotrexate, focalin,iloprost, methotrexate, nicotine, thalidomide, ambrisentan (e.g.,letairis), bosentan, atorvastatin, pentoxifylline, adalimumab,rituximab, stelara (e.g., ustekinumab), aviptadil, apremilast, remicade,humira, growth factors, chemokines, cytokines, cellular products,demethylating agents, and other factors which are now known or laterdetermined to stimulate differentiation of stem cells, insulin-likegrowth factor (IGF), platelet-derived growth factor (PDGF), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF),interleukin 18 (IL-8), an antithrombogenic agent (e.g., heparin, heparinderivatives, urokinase, or PPack (dextrophenylalanine proline argininechloromethylketone), an antithrombin compound, a platelet receptorantagonist, an anti-thrombin antibody, an anti-platelet receptorantibody, aspirin, dipyridamole, protamine, hirudin, a prostaglandininhibitor, and/or a platelet inhibitor), an antiapoptotic agent (e.g.,erythropoietin (Epo), an Epo derivative or analog, or their salts,thrombopoietin (Tpo), IGF-I, IGF-II, hepatocyte growth factor (HGF), ora caspase inhibitor), an anti-inflammatory agent (e.g., a p38 MAP kinaseinhibitor, a statin, in IL-6 inhibitor, an IL-1 inhibitor, Pemirolast,Tranilast, Remicade, Sirolimus, and/or a nonsteroidal anti-inflammatorycompound (e.g., acetylsalicylic acid, ibuprofen, Tepoxalin, Tolmetin, orSuprofen)), an immunosuppressive or immunomodulatory agent (e.g., acalcineurin inhibitor, for example cyclosporine, Tacrolimus, an mTORinhibitor such as Sirolimus or Everolimus; an anti-proliferative such asazathioprine and/or mycophenolate mofetil; a corticosteroid, e.g.,prednisone, prednisolone or hydrocortisone; a steroid-sparing agent suchas azathioprine and methotrexate; an antibody such as a monoclonalanti-IL-2Rα receptor antibody, Basiliximab, Daclizuma, polyclonalanti-T-cell antibodies such as anti-thymocyte globulin (ATG),anti-lymphocyte globulin (ALG), and/or the monoclonal anti-T cellantibody OKT3, or adherent placental stem cells as described in U.S.Pat. No. 7,468,276, and U.S. Patent Application Publication No. and2007/0275362, the disclosures of each of which are incorporated hereinby reference in their entireties), and/or an antioxidant (e.g.,probucol; vitamins A, C, and/or E, coenzyme Q-10, glutathione, Lcysteine, N-acetylcysteine, or an antioxidant derivative, analog or saltof any of the foregoing). In certain embodiments, therapeuticcompositions comprising the PDACs further comprise one or moreadditional cell types, e.g., adult cells (for example, fibroblasts orendodermal cells), stem cells and/or progenitor cells. Such therapeuticagents and/or one or more additional types of cells can be administeredto an individual in need thereof individually or in combinations or twoor more such compounds or agents.

In one embodiment, the second therapeutic agent is an anti-inflammatoryagent, steroid, immune suppressant, and/or an antibiotic. Examples ofanti-inflammatory drugs useful in the treatment of sarcoidosis orsarcoidosis-related diseases, disorders or conditions include, but arenot limited to, mesalamine, 5-ASA (5-aminosalicylic acid) agents (e.g.,ASACOL® (mesalamine, delayed-release), DIPENTUM (Osalazine), PENTASA®(mesalamine controlled-release)), sulfasalazine (a combination of 5-ASAand sulfapyridine), anti-inflammatory antibodies (e.g., Infliximab(REMICADE®)), and the like. Examples of steroids useful in the treatmentof sarcoidosis or sarcoidosis-related diseases, disorders or conditionsinclude, but are not limited to, cortisone, hydrocortisone, predisone,methylprednisone, and the like. Typically, as practiced in the art, thedosage of steroid is first delivered in a relatively large dose,followed by smaller dosages as inflammation subsides. Examples of immunesuppressants useful in the treatment of sarcoidosis orsarcoidosis-related diseases include, but are not limited to,cyclosporine A, 6-mercaptopurine or azathioprine. Any antibiotic can beused in the treatment of Crohn's disease, including, e.g., ampicillin,sulfonamide, cephalosporin, tetracycline, and/or metronidazole. Inanother specific embodiment, the second therapy is an administration ofporcine whipworms, e.g., ova of Trichuris suis.

In another embodiment, the second therapy provided herein is ananticancer therapy, e.g., to treat sarcoidosis-related cancers, e.g.,one or more chemotherapies or chemotherapeutic compounds. Such otheranticancer therapies can be administered to the individual at the sametime as, during the same course of treatment as, or separately from,said administration of placental cells, e.g., PDACs. In a specificembodiment, the administration of said other anticancer therapies isadministered sequentially with administration of said placental cells.In another specific embodiment, said other anticancer therapy oranticancer therapies are administered to said individual beforeadministration of said placental cells; e.g., a course of such otheranticancer therapies is administered to the individual, and completed,prior to administration to the individual of isolated placental cells.In another specific embodiment, said placental cells are administered tothe individual before administration of said other anticancer therapies;e.g., a course of placental cells is administered to said individualbefore administration of said other anticancer therapies, and completed,prior to administration to the individual said other anticancer therapyor anticancer therapies.

In a specific embodiment, the anticancer agent is melphalan (also knownas L-phenylalanine mustard or L-PAM; trade name Albertan). Thus, in oneembodiment, the method of treating an individual having multiple myelomacomprises administering to said individual melphalan, e.g., atherapeutically effective dose or doses of melphalan. Administration istypically oral or intravenous. In another specific embodiment, theanticancer agent is thalidomide. In another specific embodiment, theanticancer agent is an amino-substituted thalidomide analog or anamino-substituted imidazole. In another specific embodiment, theanticancer agent is pomalidomide (sold under the trade name ACTIMID®);lenalidomide (sold under the trade name REVLIMID®); or lenalidomide incombination with dexamethasone. In another specific embodiment, theanticancer treatment is bortezomib (VELCADE®). In another specificembodiment, the anticancer agent comprises a combination of melphalan,prednisone, and thalidomide (administered separately or together). Inanother specific embodiment, the anticancer agent is the combination ofbortezomib, melphalan and prednisone (administered separately ortogether).

Other anticancer agents are well-known in the art. Thus, in otherspecific embodiments, the anticancer agents include, but are not limitedto: acivicin; aclarubicin; acodazole hydrochloride; acronine;adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-aletheine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC®),imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (GENASENSE®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sd±1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

In another embodiment, the combination therapy comprises administrationof placental cells in combination with bisphosphonates. In oneembodiment, the bisphosphonates are clodronate and/or pamidronate.

In a specific embodiment, the combination therapy provided hereincomprises administration of placental cells in combination with one ormore TNF-α inhibitors (e.g., infliximab, adalimumab), corticosteroids(e.g., prednisone), PDE-4 inhibitors (e.g., apremiliast), IL-12inhibitors, IL-23 inhibitors, aviptadil, IFN-γ inhibitors, orantimalarials (e.g., chloroquine or hydroxychloroquine).

In a specific embodiment, said therapeutically effective amount ofplacental cells, optionally in combination with one or more of a secondtherapy (e.g., second therapeutic agent), reduces, e.g., detectablyreduces, the number of, or degree of severity of, or reduces the rate ofincrease in the number of, or degree of severity, one or more granulomasin said individual.

In another specific embodiment, said therapeutically effective amount ofplacental cells, optionally in combination with one or more of a secondtherapy (e.g., second therapeutic agent), reduces, e.g., detectablyreduces, the number of, or reduces the rate of increase in the numberof, monocytes, macrophages, or activated T-lymphocytes, in combinationwith one or more of a second therapy (e.g., second therapeutic agent).In another specific embodiment, said therapeutically effective amount ofplacental cells, optionally in combination with one or more of a secondtherapy (e.g., second therapeutic agent), reduces, e.g., detectablyreduces, the level of, or reduces the rate of increase in the level of,one or more TNF-α, IL-2, IL-12, IFN-γ, IL-1, IL-6, IL-15, or Th1response in said individual, in combination with one or more of a secondtherapy (e.g., second therapeutic agent).

4.2 Isolated Placental Cells and Isolated Placental Cell Populations

The isolated placental cells, e.g., PDACs, useful in the treatment ofindividuals having sarcoidosis or a sarcoidosis-related disease ordisorder, are cells, obtainable from a placenta or part thereof, thatadhere to a tissue culture substrate and have characteristics ofmultipotent cells or stem cells, but are not trophoblasts. In certainembodiments, the isolated placental cells useful in the methodsdisclosed herein have the capacity to differentiate into non-placentalcell types.

The isolated placental cells useful in the methods disclosed herein canbe either fetal or maternal in origin (that is, can have the genotype ofeither the fetus or mother, respectively). Preferably, the isolatedplacental cells and populations of isolated placental cells are fetal inorigin. As used herein, the phrase “fetal in origin” or “non-maternal inorigin” indicates that the isolated placental cells or populations ofisolated placental cells are obtained from the umbilical cord orplacental structures associated with the fetus, i.e., that have thefetal genotype. As used herein, the phrase “maternal in origin”indicates that the cells or populations of cells are obtained from aplacental structures associated with the mother, e.g., which have thematernal genotype. Isolated placental cells, e.g., PDACs, or populationsof cells comprising the isolated placental cells, can comprise isolatedplacental cells that are solely fetal or maternal in origin, or cancomprise a mixed population of isolated placental cells of both fetaland maternal origin. The isolated placental cells, and populations ofcells comprising the isolated placental cells, can be identified andselected by the morphological, marker, and culture characteristicsdiscussed below. In certain embodiments, any of the placental cells,e.g., placental stem cells or placental multipotent cells describedherein, are autologous to a recipient, e.g., an individual who hassarcoidosis or a sarcoidosis-related disease or disorder. In certainother embodiments, any of the placental cells, e.g., placental stemcells or placental multipotent cells described herein, are heterologousto a recipient, e.g., an individual who has sarcoidosis or asarcoidosis-related disease or disorder.

4.2.1 Physical and Morphological Characteristics

The isolated placental cells described herein (PDACs), when cultured inprimary cultures or in cell culture, adhere to the tissue culturesubstrate, e.g., tissue culture container surface (e.g., tissue cultureplastic), or to a tissue culture surface coated with extracellularmatrix or ligands such as laminin, collagen (e.g., native or denatured),gelatin, fibronectin, ornithine, vitronectin, and extracellular membraneprotein (e.g., MATRIGEL® (BD Discovery Labware, Bedford, Mass.)). Theisolated placental cells in culture assume a generally fibroblastoid,stellate appearance, with a number of cytoplasmic processes extendingfrom the central cell body. The cells are, however, morphologicallydistinguishable from fibroblasts cultured under the same conditions, asthe isolated placental cells exhibit a greater number of such processesthan do fibroblasts. Morphologically, isolated placental cells are alsodistinguishable from hematopoietic stem cells, which generally assume amore rounded, or cobblestone, morphology in culture.

In certain embodiments, the isolated placental cells useful in themethods disclosed herein, when cultured in a growth medium, developembryoid-like bodies. Embryoid-like bodies are noncontiguous clumps ofcells that can grow on top of an adherent layer of proliferatingisolated placental cells. The term “embryoid-like” is used because theclumps of cells resemble embryoid bodies, clumps of cells that grow fromcultures of embryonic stem cells. Growth medium in which embryoid-likebodies can develop in a proliferating culture of isolated placentalcells includes medium comprising, e.g., DMEM-LG (e.g., from Gibco); 2%fetal calf serum (e.g., from Hyclone Labs.); 1×insulin-transferrin-selenium (ITS); 1× linoleic acid-bovine serumalbumin (LA-BSA); 10⁻⁹ M dexamethasone (e.g., from Sigma); 10⁻⁴ Mascorbic acid 2-phosphate (e.g., from Sigma); epidermal growth factor 10ng/mL (e.g., from R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (e.g., from R&D Systems).

4.2.2 Cell Surface, Molecular and Genetic Markers

The isolated placental cells, e.g., isolated multipotent placental cellsor isolated placental stem cells, and populations of such isolatedplacental cells, useful in the methods of disclosed herein, e.g., themethods of treatment of sarcoidosis or a sarcoidosis-related disease ordisorder, are tissue culture plastic-adherent human placental cells thathave characteristics of multipotent cells or stem cells, and express aplurality of markers that can be used to identify and/or isolate thecells, or populations of cells that comprise the stem cells. In certainembodiments, the PDACs are angiogenic, e.g., in vitro or in vivo. Theisolated placental cells, and placental cell populations describedherein (that is, two or more isolated placental cells) include placentalcells and placental cell-containing cell populations-obtained directlyfrom the placenta, or any part thereof (e.g., chorion, placentalcotyledons, or the like). Isolated placental cell populations alsoinclude populations of (that is, two or more) isolated placental cellsin culture, and a population in a container, e.g., a bag. The isolatedplacental cells described herein are not bone marrow-derived mesenchymalcells, adipose-derived mesenchymal stem cells, or mesenchymal cellsobtained from umbilical cord blood, placental blood, or peripheralblood. Placental cells, e.g., placental multipotent cells and placentalcells, useful in the methods and compositions described herein aredescribed herein and, e.g., in U.S. Pat. Nos. 7,311,904; 7,311,905; and7,468,276; and in U.S. Patent Application Publication No. 2007/0275362,the disclosures of which are hereby incorporated by reference in theirentireties.

In certain embodiments, the isolated placental cells are isolatedplacental stem cells. In certain other embodiments, the isolatedplacental cells are isolated placental multipotent cells. In oneembodiment, the isolated placental cells, e.g, PDACs, are CD34⁻, CD10⁺and CD105⁺ as detected by flow cytometry. In another specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells have thepotential to differentiate into cells of a neural phenotype, cells of anosteogenic phenotype, and/or cells of a chondrogenic phenotype. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells are additionally CD200⁺. In another specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ placental cells are additionally CD45⁻ orCD90⁺. In another specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺placental cells are additionally CD45⁻ and CD90⁺, as detected by flowcytometry. In another specific embodiment, the isolated CD34⁻, CD10⁺,CD105⁺, CD200⁺ placental cells are additionally CD90⁺ or CD45⁻, asdetected by flow cytometry. In another specific embodiment, the isolatedCD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells are additionally CD90⁺ andCD45⁻, as detected by flow cytometry, i.e., the cells are CD34⁻, CD10⁺,CD45⁻, CD90⁺, CD105⁺ and CD200⁺. In another specific embodiment, saidCD34⁻, CD10⁺, CD45⁻, CD90⁺, CD105⁺, CD200⁺ cells are additionally CD80⁻and CD86⁻.

In certain embodiments, said placental cells are CD34⁻, CD10⁺, CD105⁺and CD200⁺, and one or more of CD38⁻, CD45⁻, CD80⁻, CD86⁻, CD133⁻,HLA-DR,DP,DQ⁻, SSEA3⁻, SSEA4⁻, CD29⁺, CD44⁺, CD73⁺, CD90⁺, CD105⁺,HLA-A,B,C⁺, PDL1⁺, ABC-p⁺, and/or OCT-4⁺, as detected by flow cytometry.In other embodiments, any of the CD34⁻, CD10⁺, CD105⁺ cells describedabove are additionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, SH3⁺or SH4⁺. In another specific embodiment, the cells are additionallyCD44⁺. In another specific embodiment of any of the isolated CD34⁻,CD10⁺, CD105⁺ placental cells above, the cells are additionally one ormore of CD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, orProgrammed Death-1 Ligand (PDL1)⁺, or any combination thereof.

In another embodiment, the CD34⁻, CD10⁺, CD105⁺ cells are additionallyone or more of CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD62E⁻,CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺, SH2⁺(CD105⁺), CD106/VCAM⁺, CD117⁻, CD144/VE-cadherin^(low), CD184/CXCR4⁻,CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻),HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, or Programmed Death-1 Ligand (PDL1)⁺,or any combination thereof. In a other embodiment, the CD34⁻, CD10⁺,CD105⁺ cells are additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻,CD54/ICAM⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻,CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR⁴⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, andProgrammed Death-1 Ligand (PDL1)⁺.

In another specific embodiment, any of the placental cells describedherein are additionally ABC-p⁺, as detected by flow cytometry, or OCT-4⁺(POU5F1⁺), as determined by RT-PCR, wherein ABC-p is a placenta-specificABC transporter protein (also known as breast cancer resistance protein(BCRP) and as mitoxantrone resistance protein (MXR)), and OCT-4 is theOctamer-4 protein (POU5F1). In another specific embodiment, any of theplacental cells described herein are additionally SSEA3⁻ or SSEA4⁻, asdetermined by flow cytometry, wherein SSEA3 is Stage Specific EmbryonicAntigen 3, and SSEA4 is Stage Specific Embryonic Antigen 4. In anotherspecific embodiment, any of the placental cells described herein areadditionally SSEA3⁻ and SSEA4⁻.

In another specific embodiment, any of the placental cells describedherein are additionally one or more of MHC-I⁺ (e.g., HLA-A,B,C⁺),MHC-II⁻ (e.g., HLA-DP,DQ,DR⁻) or HLA-G⁻. In another specific embodiment,any of the placental cells described herein are additionally one or moreof MHC-I⁺ (e.g., HLA-A,B,C⁺), MHC-II⁻ (e.g., HLA-DP,DQ,DR⁻) and HLA-G⁻.

Also provided herein are populations of the isolated placental cells, orpopulations of cells, e.g., populations of placental cells, comprising,e.g., that are enriched for, the isolated placental cells, that areuseful in the methods and compositions disclosed herein. Preferredpopulations of cells comprising the isolated placental cells, whereinthe populations of cells comprise, e.g., at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or98% isolated CD10⁺, CD105⁺ and CD34⁻ placental cells; that is, at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 98% of cells in said population are isolatedCD10⁺, CD105⁺ and CD34⁻ placental cells. In a specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ placental cells are additionally CD200⁺.In another specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺,CD200⁺ placental cells are additionally CD90⁺ or CD45⁻, as detected byflow cytometry. In another specific embodiment, the isolated CD34⁻,CD10⁺, CD105⁺, CD200⁺ placental cells are additionally CD90⁺ and CD45⁻,as detected by flow cytometry. In another specific embodiment, any ofthe isolated CD34⁻, CD10⁺, CD105⁺ placental cells described above areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, SH3⁺ or SH4⁺. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells, or isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ placental cells, areadditionally CD44⁺. In a specific embodiment of any of the populationsof cells comprising isolated CD34⁻, CD10⁺, CD105⁺ placental cells above,the isolated placental cells are additionally one or more of CD13⁺,CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺(CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺,CD117⁻, CD144/VE-cadherin^(low), CD184/CXCR⁴⁻, CD200⁺, CD133⁻, OCT-4⁺,SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻,HLA-G⁻, or Programmed Death-1 Ligand (PDL1)⁺, or any combinationthereof. In another specific embodiment, the CD34⁻, CD10⁺, CD105⁺ cellsare additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54/ICAM⁺,CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺,SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻, CD144/VE-cadherin^(low),CD184/CXCR⁴⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻(VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, and Programmed Death-1Ligand (PDL1)⁺.

In certain embodiments, the isolated placental cells useful in themethods and compositions described herein are one or more, or all, ofCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺,SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, and ABC-p⁺, wherein said isolatedplacental cells are obtained by physical and/or enzymatic disruption ofplacental tissue. In a specific embodiment, the isolated placental cellsare OCT-4⁺ and ABC-p⁺. In another specific embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, wherein said isolated placentalcells have at least one of the following characteristics: CD10⁺, CD29⁺,CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH3⁺, SH4⁺, SSEA3⁻, and SSEA4⁻. In anotherspecific embodiment, the isolated placental cells are OCT-4⁺, CD34⁻,CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH3⁺, SH4⁺, SSEA3⁻, andSSEA4⁻. In another embodiment, the isolated placental cells are OCT-4⁺,CD34⁻, SSEA3⁻, and SSEA4⁻. In another specific embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, and is either SH2⁺ or SH3⁺. Inanother specific embodiment, the isolated placental cells are OCT-4⁺,CD34⁻, SH2⁺, and SH3⁺. In another specific embodiment, the isolatedplacental cells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻, and are eitherSH2⁺ or SH3⁺. In another specific embodiment, the isolated placentalcells are OCT-4⁺ and CD34⁻, and either SH2⁺ or SH3⁺, and is at least oneof CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, or SSEA4⁻. Inanother specific embodiment, the isolated placental cells are OCT-4⁺,CD34⁻, CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, and SSEA4⁻, andeither SH2⁺ or SH3⁺.

In another embodiment, the isolated placental cells useful in themethods and compositions disclosed herein are SH2⁺, SH3⁺, SH4⁺ andOCT-4⁺. In another specific embodiment, the isolated placental cells areCD10⁺, CD29⁺, CD44⁺, CD54⁺, CD90⁺, CD34⁻, CD45⁻, SSEA3⁻, or SSEA4⁻. Inanother embodiment, the isolated placental cells are SH2⁺, SH3⁺, SH4⁺,SSEA3⁻ and SSEA4⁻. In another specific embodiment, the isolatedplacental cells are SH2⁺, SH3⁺, SH4⁺, SSEA3⁻ and SSEA4⁻, CD10⁺, CD29⁺,CD44⁺, CD54⁺, CD90⁺, OCT-4⁺, CD34⁻ or CD45⁻.

In another embodiment, the isolated placental cells useful in themethods and compositions disclosed herein are CD10⁺, CD29⁺ ^(,) CD34,CD44⁺ ^(,) CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, and SH4⁺; wherein saidisolated placental cells are additionally one or more of OCT-4⁺, SSEA3⁻or SSEA4⁻.

In certain embodiments, isolated placental cells useful in the methodsand compositions disclosed herein are CD200⁺ or HLA-G⁻. In a specificembodiment, the isolated placental cells are CD200⁺ and HLA-G⁻. Inanother specific embodiment, the isolated placental cells areadditionally CD73⁺ and CD105⁺. In another specific embodiment, theisolated placental cells are additionally CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, the isolated placental cells areadditionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In anotherspecific embodiment, said isolated CD200⁺ or HLA-G⁻ placental cellsfacilitate the formation of embryoid-like bodies in a population ofplacental cells comprising the isolated placental cells, underconditions that allow the formation of embryoid-like bodies. In anotherspecific embodiment, the isolated placental cells are isolated away fromplacental cells that are not stem or multipotent cells. In anotherspecific embodiment, said isolated placental cells are isolated awayfrom placental cells that do not display these markers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, CD200⁺, HLA-G⁻ stem cells. In a specificembodiment, said population is a population of placental cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said cell population are isolated CD200⁺, HLA-G⁻ placentalcells. Preferably, at least about 70% of cells in said cell populationare isolated CD200⁺, HLA-G⁻ placental cells. More preferably, at leastabout 90%, 95%, or 99% of said cells are isolated CD200⁺, HLA-G⁻placental cells. In a specific embodiment of the cell populations, saidisolated CD200⁺, HLA-G⁻ placental cells are also CD73⁺ and CD105⁺. Inanother specific embodiment, said isolated CD200⁺, HLA-G⁻ placentalcells are also CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment,said isolated CD200⁺, HLA-G⁻ placental cells are also CD34⁻, CD38⁻,CD45⁻, CD73⁺ and CD105⁺. In another embodiment, said cell populationproduces one or more embryoid-like bodies when cultured under conditionsthat allow the formation of embryoid-like bodies. In another specificembodiment, said cell population is isolated away from placental cellsthat are not stem cells. In another specific embodiment, said isolatedCD200⁺, HLA-G⁻ placental cells are isolated away from placental cellsthat do not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are CD73⁺, CD105⁺, and CD200⁺.In another specific embodiment, the isolated placental cells are HLA-G⁻.In another specific embodiment, the isolated placental cells are CD34⁻,CD38⁻ or CD45⁻. In another specific embodiment, the isolated placentalcells are CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, theisolated placental cells are CD34⁻, CD38⁻, CD45⁻, and HLA-G⁻. In anotherspecific embodiment, the isolated CD73⁺, CD105⁺, and CD200⁺ placentalcells facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising the isolated placental cells,when the population is cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, theisolated placental cells are isolated away from placental cells that arenot the isolated placental cells. In another specific embodiment, theisolated placental cells are isolated away from placental cells that donot display these markers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated CD73⁺, CD105⁺, CD200⁺ placental cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said cell population are isolated CD73⁺, CD105⁺, CD200⁺placental cells. In another embodiment, at least about 70% of said cellsin said population of cells are isolated CD73⁺, CD105⁺, CD200⁺ placentalcells. In another embodiment, at least about 90%, 95% or 99% of cells insaid population of cells are isolated CD73⁺, CD105⁺, CD200⁺ placentalcells. In a specific embodiment of said populations, the isolatedplacental cells are HLA-G⁻. In another specific embodiment, the isolatedplacental cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, the isolated placental cells are additionallyCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, the isolatedplacental cells are additionally CD34⁻, CD38⁻, CD45⁻, and HLA-G⁻. Inanother specific embodiment, said population of cells produces one ormore embryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidpopulation of placental cells is isolated away from placental cells thatare not stem cells. In another specific embodiment, said population ofplacental cells is isolated away from placental cells that do notdisplay these characteristics.

In certain other embodiments, the isolated placental cells are one ormore of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, SH4⁺, SSEA3-, SSEA4⁻, OCT-4⁺, HLA-G⁻ or ABC-p⁺. In a specificembodiment, the isolated placental cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻,CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3-, SSEA4⁻, andOCT-4⁺. In another specific embodiment, the isolated placental cells areCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺, SH3⁺, and SH4⁺. Inanother specific embodiment, the isolated placental cells are CD10⁺,CD29⁺, CD34⁻, CD38⁻, CD45⁻, CD54⁺, SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. Inanother specific embodiment, the isolated placental cells are CD10⁺,CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, HLA-G⁻, SH2⁺, SH3⁺,SH4⁺. In another specific embodiment, the isolated placental cells areOCT-4⁺ and ABC-p⁺. In another specific embodiment, the isolatedplacental cells are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. In another embodiment,the isolated placental cells are OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻. In aspecific embodiment, said isolated OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻placental cells are additionally CD10⁺, CD29⁺, CD34⁻, CD44⁺, CD45⁻,CD54⁺, CD90⁺, SH2⁺, SH3⁺, and SH4⁺. In another embodiment, the isolatedplacental cells are OCT-4⁺ and CD34⁻, and either SH3⁺ or SH4⁺. Inanother embodiment, the isolated placental cells are CD34⁻ and eitherCD10⁺, CD29⁺, CD44⁺, CD54⁺, CD90⁺, or OCT-4⁺.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are CD200⁺ and OCT-4⁺. In aspecific embodiment, the isolated placental cells are CD73⁺ and CD105⁺.In another specific embodiment, said isolated placental cells areHLA-G⁻. In another specific embodiment, said isolated CD200⁺, OCT-4⁺placental cells are CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said isolated CD200⁺, OCT-4⁺ placental cells are CD34⁻,CD38⁻ and CD45⁻. In another specific embodiment, said isolated CD200⁺,OCT-4⁺ placental cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁻. In another specific embodiment, the isolated CD200⁺, OCT-4⁺placental cells facilitate the production of one or more embryoid-likebodies by a population of placental cells that comprises the isolatedcells, when the population is cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidisolated CD200⁺, OCT-4⁺ placental cells are isolated away from placentalcells that are not stem cells. In another specific embodiment, saidisolated CD200⁺, OCT-4⁺ placental cells are isolated away from placentalcells that do not display these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, CD200⁺, OCT-4⁺ placental cells. In variousembodiments, at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, or at least about 60% of cellsin said cell population are isolated CD200⁺, OCT-4⁺ placental cells. Inanother embodiment, at least about 70% of said cells are said isolatedCD200⁺, OCT-4⁺ placental cells. In another embodiment, at least about80%, 90%, 95%, or 99% of cells in said cell population are said isolatedCD200⁺, OCT-4⁺ placental cells. In a specific embodiment of the isolatedpopulations, said isolated CD200⁺, OCT-4⁺ placental cells areadditionally CD73⁺ and CD105⁺. In another specific embodiment, saidisolated CD200⁺, OCT-4⁺ placental cells are additionally HLA-G⁻. Inanother specific embodiment, said isolated CD200⁺, OCT-4⁺ placentalcells are additionally CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said isolated CD200⁺, OCT-4⁺ placental cells areadditionally CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁻. In anotherspecific embodiment, the cell population produces one or moreembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidcell population is isolated away from placental cells that are notisolated CD200⁺, OCT-4⁺ placental cells. In another specific embodiment,said cell population is isolated away from placental cells that do notdisplay these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are CD73⁺, CD105⁺ and HLA-G⁻.In another specific embodiment, the isolated CD73⁺, CD105⁺ and HLA-G⁻placental cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ placental cellsare additionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,the isolated CD73⁺, CD105⁺, HLA-G⁻ placental cells are additionallyOCT-4⁺. In another specific embodiment, the isolated CD73⁺, CD105⁺,HLA-G⁻ placental cells are additionally CD200⁺. In another specificembodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ placental cells areadditionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specificembodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ placental cellsfacilitate the formation of embryoid-like bodies in a population ofplacental cells comprising said cells, when the population is culturedunder conditions that allow the formation of embryoid-like bodies. Inanother specific embodiment, said the isolated CD73⁺, CD105⁺, HLA-G⁻placental cells are isolated away from placental cells that are not theisolated CD73⁺, CD105⁺, HLA-G⁻ placental cells. In another specificembodiment, said the isolated CD73⁺, CD105⁺, HLA-G⁻ placental cells areisolated away from placental cells that do not display these markers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated CD73⁺, CD105⁺ and HLA-G⁻ placental cells.In various embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said population of cells are isolated CD73⁺, CD105⁺, HLA-G⁻placental cells. In another embodiment, at least about 70% of cells insaid population of cells are isolated CD73⁺, CD105⁺, HLA-G⁻ placentalcells. In another embodiment, at least about 90%, 95% or 99% of cells insaid population of cells are isolated CD73⁺, CD105⁺, HLA-G⁻ placentalcells. In a specific embodiment of the above populations, said isolatedCD73⁺, CD105⁺, HLA-G⁻ placental cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺,HLA-G⁻ placental cells are additionally CD34⁻, CD38⁻ and CD45⁻. Inanother specific embodiment, said isolated CD73⁺, CD105⁺, HLA-G⁻placental cells are additionally OCT-4⁺. In another specific embodiment,said isolated CD73⁺, CD105⁺, HLA-G⁻ placental cells are additionallyCD200⁺. In another specific embodiment, said isolated CD73⁺, CD105⁺,HLA-G⁻ placental cells are additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said cell population is isolatedaway from placental cells that are not CD73⁺, CD105⁺, HLA-G⁻ placentalcells. In another specific embodiment, said cell population is isolatedaway from placental cells that do not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are CD73⁺ and CD105⁺ andfacilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said CD73⁺, CD105⁺cells when said population is cultured under conditions that allowformation of embryoid-like bodies. In another specific embodiment, saidisolated CD73⁺, CD105⁺ placental cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are additionally CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said isolated CD73⁺, CD105⁺ placental cells areadditionally OCT-4⁺. In another specific embodiment, said isolatedCD73⁺, CD105⁺ placental cells are additionally OCT-4⁺, CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are isolated away from placental cells that are not saidcells. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are isolated away from placental cells that do notdisplay these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated placental cells that are CD73⁺, CD105⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said cells when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells are saidisolated CD73⁺, CD105⁺ placental cells. In another embodiment, at leastabout 70% of cells in said population of cells are said isolated CD73⁺,CD105⁺ placental cells. In another embodiment, at least about 90%, 95%or 99% of cells in said population of cells are said isolated CD73⁺,CD105⁺ placental cells. In a specific embodiment of the abovepopulations, said isolated CD73⁺, CD105⁺ placental cells areadditionally CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidisolated CD73⁺, CD105⁺ placental cells are additionally CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺placental cells are additionally OCT-4⁺. In another specific embodiment,said isolated CD73⁺, CD105⁺ placental cells are additionally CD200⁺. Inanother specific embodiment, said isolated CD73⁺, CD105⁺ placental cellsare additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In anotherspecific embodiment, said cell population is isolated away fromplacental cells that are not said isolated CD73⁺, CD105⁺ placentalcells. In another specific embodiment, said cell population is isolatedaway from placental cells that do not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are OCT-4⁺ and facilitateformation of one or more embryoid-like bodies in a population ofisolated placental cells comprising said cells when cultured underconditions that allow formation of embryoid-like bodies. In a specificembodiment, said isolated OCT-4⁺ placental cells are additionally CD73⁺and CD105⁺. In another specific embodiment, said isolated OCT-4⁺placental cells are additionally CD34⁻, CD38⁻, or CD45⁻. In anotherspecific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD200⁺. In another specific embodiment, said isolatedOCT-4⁺ placental cells are additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻,CD38⁻, and CD45⁻. In another specific embodiment, said isolated OCT-4⁺placental cells are isolated away from placental cells that are notOCT-4⁺ placental cells. In another specific embodiment, said isolatedOCT-4⁺ placental cells are isolated away from placental cells that donot display these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated placental cells that are OCT-4⁺ andfacilitate the formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said cells when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells are saidisolated OCT-4⁺ placental cells. In another embodiment, at least about70% of cells in said population of cells are said isolated OCT-4⁺placental cells. In another embodiment, at least about 80%, 90%, 95% or99% of cells in said population of cells are said isolated OCT-4⁺placental cells. In a specific embodiment of the above populations, saidisolated OCT-4⁺ placental cells are additionally CD34⁻, CD38⁻ or CD45⁻.In another specific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said isolated OCT-4⁺ placental cells are additionally CD73⁺ and CD105⁺.In another specific embodiment, said isolated OCT-4⁺ placental cells areadditionally CD200⁺. In another specific embodiment, said isolatedOCT-4⁺ placental cells are additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻,CD38⁻, and CD45⁻. In another specific embodiment, said cell populationis isolated away from placental cells that are not said cells. Inanother specific embodiment, said cell population is isolated away fromplacental cells that do not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated HLA-A,B,C⁺,CD45⁻, CD133⁻ and CD34⁻ placental cells. In another embodiment, a cellpopulation useful in the methods and compositions described herein is apopulation of cells comprising isolated placental cells, wherein atleast about 70%, at least about 80%, at least about 90%, at least about95% or at least about 99% of cells in said isolated population of cellsare isolated HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻ placental cells. In aspecific embodiment, said isolated placental cell or population ofisolated placental cells is isolated away from placental cells that arenot HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻ placental cells. In anotherspecific embodiment, said isolated placental cells are non-maternal inorigin. In another specific embodiment, said isolated population ofplacental cells are substantially free of maternal components; e.g., atleast about 40%, 45%, 5-0%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%,98% or 99% of said cells in said isolated population of placental cellsare non-maternal in origin.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated CD10⁺, CD13⁺,CD33⁺, CD45⁻, CD117⁻ and CD133⁻ placental cells. In another embodiment,a cell population useful in the methods and compositions describedherein is a population of cells comprising isolated placental cells,wherein at least about 70%, at least about 80%, at least about 90%, atleast about 95% or at least about 99% of cells in said population ofcells are isolated CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD11T and CD133⁻placental cells. In a specific embodiment, said isolated placental cellsor population of isolated placental cells is isolated away fromplacental cells that are not said isolated placental cells. In anotherspecific embodiment, said isolated CD10⁺, CD13⁺, CD33⁺, CD45⁻, CD117⁻and CD133⁻ placental cells are non-maternal in origin, i.e., have thefetal genotype. In another specific embodiment, at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of saidcells in said isolated population of placental cells, are non-maternalin origin. In another specific embodiment, said isolated placental cellsor population of isolated placental cells are isolated away fromplacental cells that do not display these characteristics.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated CD10⁻, CD33⁻,CD44⁺, CD45⁻, and CD11T placental cells. In another embodiment, a cellpopulation useful for the in the methods and compositions describedherein is a population of cells comprising, e.g., enriched for, isolatedplacental cells, wherein at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 99% of cells insaid population of cells are isolated CD10⁻, CD33⁻, CD44⁺, CD45⁻, andCD11T placental cells. In a specific embodiment, said isolated placentalcell or population of isolated placental cells is isolated away fromplacental cells that are not said cells. In another specific embodiment,said isolated placental cells are non-maternal in origin. In anotherspecific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cellpopulation are non-maternal in origin. In another specific embodiment,said isolated placental cell or population of isolated placental cellsis isolated away from placental cells that do not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated CD10⁻, CD13⁻,CD33⁻, CD45⁻, and CD117⁻ placental cells. In another embodiment, a cellpopulation useful for in the methods and compositions described hereinis a population of cells comprising, e.g., enriched for, isolated CD10⁻,CD13⁻, CD33⁻, CD45⁻, and CD117⁻ placental cells, wherein at least about70%, at least about 80%, at least about 90%, at least about 95% or atleast about 99% of cells in said population are CD10⁻, CD13⁻, CD33⁻,CD45⁻, and CD117⁻ placental cells. In a specific embodiment, saidisolated placental cells or population of isolated placental cells areisolated away from placental cells that are not said cells. In anotherspecific embodiment, said isolated placental cells are non-maternal inorigin. In another specific embodiment, at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells insaid cell population are non-maternal in origin. In another specificembodiment, said isolated placental cells or population of isolatedplacental cells is isolated away from placental cells that do notdisplay these characteristics.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are HLA A,B,C⁺, CD45⁻, CD34⁻,and CD133⁻, and are additionally CD10⁺, CD13⁺, CD38⁺, CD44⁺, CD90⁺,CD105⁺, CD200⁺ and/or HLA-G⁻, and/or negative for CD117. In anotherembodiment, a cell population useful in the methods described herein isa population of cells comprising isolated placental cells, wherein atleast about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98% or about 99% of the cells in said population areisolated placental cells that are HLA A,B,C⁻, CD45⁻, CD34⁻, CD133⁻, andthat are additionally positive for CD10, CD13, CD38, CD44, CD90, CD105,CD200, and/or negative for CD117 and/or HLA-G. In a specific embodiment,said isolated placental cells or population of isolated placental cellsare isolated away from placental cells that are not said cells. Inanother specific embodiment, said isolated placental cells arenon-maternal in origin. In another specific embodiment, at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99%of said cells in said cell population are non-maternal in origin. Inanother specific embodiment, said isolated placental cells or populationof isolated placental cells are isolated away from placental cells thatdo not display these markers.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated placental cellsthat are CD200⁺ and CD10⁺, as determined by antibody binding, andCD117⁻, as determined by both antibody binding and RT-PCR. In anotherembodiment, the isolated placental cells useful in the methods andcompositions described herein are isolated placental cells, e.g.,placental stem cells or placental multipotent cells, that are CD10⁺,CD29⁻, CD54⁺, CD200⁺, HLA-G⁻, MHC class I⁺ and β-2-microglobulin⁺. Inanother embodiment, isolated placental cells useful in the methods andcompositions described herein are placental cells wherein the expressionof at least one cellular marker is at least two-fold higher than for amesenchymal stem cell (e.g., a bone marrow-derived mesenchymal stemcell). In another specific embodiment, said isolated placental cells arenon-maternal in origin. In another specific embodiment, at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99%of said cells in said cell population are non-maternal in origin.

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated placental cells,e.g., placental stem cells or placental multipotent cells, that are oneor more of CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62E⁻, CD62L⁻,CD62P⁻, CD80⁻, CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺,CD144/VE-cadherin^(low), CD184/CXCR4⁻, β2-microglobulin^(low),MHC-I^(low), MHC-II⁻, HLA-G^(low), and/or PDL1^(low). In a specificembodiment, the isolated placental cells are at least CD29⁺ and CD54⁺.In another specific embodiment, the isolated placental cells are atleast CD44⁺ and CD106⁺. In another specific embodiment, the isolatedplacental cells are at least CD29⁺.

In another embodiment, a cell population useful in the methods andcompositions described herein comprises isolated placental cells, and atleast 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said cellpopulation are isolated placental cells that are one or more of CD10⁺,CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻, CD62-P⁻, CD80⁻,CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(dim),CD184/CXCR4⁻, β2-microglobulin^(dim), HLA-I^(dim), HLA-II⁻, HLA-G^(dim),and/or PDL1^(dim). In another specific embodiment, at least 50%, 60%,70%, 80%, 90%, 95%, 98% or 99% of cells in said cell population areCD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻, CD62-P⁻,CD80⁻, CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺,CD144/VE-cadherin^(dim), CD184/CXCR4⁻, β2-microglobulin^(dim),MHC-I^(dim), MHC-II⁻, HLA-G^(dim), and PDL1^(dim).

In another embodiment, the isolated placental cells useful in themethods and compositions described herein are isolated placental cellsthat are one or more, or all, of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺,CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3⁻, SSEA4⁻, OCT-4⁺, andABC-p⁺, where ABC-p is a placenta-specific ABC transporter protein (alsoknown as breast cancer resistance protein (BCRP) and as mitoxantroneresistance protein (MXR)), wherein said isolated placental cells areobtained by perfusion of a mammalian, e.g., human, placenta that hasbeen drained of cord blood and perfused to remove residual blood.

In another specific embodiment of any of the above characteristics,expression of the cellular marker (e.g., cluster of differentiation orimmunogenic marker) is determined by flow cytometry; in another specificembodiment, expression of the marker is determined by RT-PCR.

Gene profiling confirms that isolated placental cells, and populationsof isolated placental cells, are distinguishable from other cells, e.g.,mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells. The isolated placental cells described herein can bedistinguished from, e.g., mesenchymal stem cells on the basis of theexpression of one or more genes, the expression of which issignificantly higher in the isolated placental cells, or in certainisolated umbilical cord stem cells, in comparison to bone marrow-derivedmesenchymal stem cells. In particular, the isolated placental cells,useful in the methods of treatment provided herein, can be distinguishedfrom mesenchymal stem cells on the basis of the expression of one ormore genes, the expression of which is significantly higher (that is, atleast twofold higher) in the isolated placental cells than in anequivalent number of bone marrow-derived mesenchymal stem cells, whereinthe one or more genes are ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE,C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1,FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6,IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7,PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21,TGFB2, VTN, ZC3H12A, or a combination of any of the foregoing, when thecells are grown under equivalent conditions. See, e.g., U.S. PatentApplication Publication No. 2007/0275362, the disclosure of which isincorporated herein by reference in its entirety. In certain specificembodiments, said expression of said one ore more genes is determined,e.g., by RT-PCR or microarray analysis, e.g, using a U133-A microarray(Affymetrix). In another specific embodiment, said isolated placentalcells express said one or more genes when cultured for a number ofpopulation doublings, e.g., anywhere from about 3 to about 35 populationdoublings, in a medium comprising DMEM-LG (e.g., from Gibco); 2% fetalcalf serum (e.g., from Hyclone Labs.); 1× insulin-transferrin-selenium(ITS); 1× linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹ Mdexamethasone (e.g., from Sigma); 10⁻⁴ M ascorbic acid 2-phosphate(e.g., from Sigma); epidermal growth factor 10 ng/mL (e.g., from R&DSystems); and platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g.,from R&D Systems). In another specific embodiment, the isolatedplacental cell-specific or isolated umbilical cord cell-specific gene isCD200.

Specific sequences for these genes can be found in GenBank at accessionnos. NM_(—)001615 (ACTG2), BC065545 (ADARB1), (NM_(—)181847 (AMIGO2),AY358590 (ARTS-1), BC074884 (B4GALT6), BC008396 (BCHE), BC020196(C11orf9), BC031103 (CD200), NM_(—)001845 (COL4A1), NM_(—)001846(COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (DSC3), NM_(—)001943(DSG2), AF338241 (ELOVL2), AY336105 (F2RL1), NM_(—)018215 (FLJ10781),AY416799 (GATA6), BC075798 (GPR126), NM_(—)016235 (GPRC5B), AF340038(ICAM1), BC000844 (IER3), BC066339 (IGFBP7), BC013142 BT019749 (IL6),BC007461 (IL18), (BC072017) KRT18, BC075839 (KRT8), BC060825 (LIPG),BC065240 (LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3),NM_(—)014840 (NUAK1), AB006755 (PCDH7), NM_(—)014476 (PDLIM3), BC126199(PKP-2), BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6),BC001201 (ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21),BC096235 (TGFB2), BC005046 (VTN), and BC005001 (ZC3H12A) as of March2008.

In certain specific embodiments, said isolated placental cells expresseach of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9, CD200,COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, F1110781, GATA6,GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18,KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1,SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12Aat a detectably higher level than an equivalent number of bonemarrow-derived mesenchymal stem cells, when the cells are grown underequivalent conditions.

In specific embodiments, the placental cells express CD200 and ARTS1(aminopeptidase regulator of type 1 tumor necrosis factor); ARTS-1 andLRAP (leukocyte-derived arginine aminopeptidase); IL6 (interleukin-6)and TGFB2 (transforming growth factor, beta 2); IL6 and KRT18 (keratin18); IER3 (immediate early response 3), MEST (mesoderm specifictranscript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200 andKRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3 (nuclear factor(erythroid-derived 2)-like 3); or CD200 and TGFB2 at a detectably higherlevel than an equivalent number of bone marrow-derived mesenchymal stemcells (BM-MSCs) wherein said bone marrow-derived mesenchymal stem cellshave undergone a number of passages in culture equivalent to the numberof passages said isolated placental cells have undergone. In otherspecific embodiments, the placental cells express ARTS-1, CD200, IL6 andLRAP; ARTS-1, IL6, TGFB2, IER3, KRT18 and MEST; CD200, IER3, IL6, KRT18,LRAP, MEST, NFE2L3, and TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP,MEST, NFE2L3, and TGFB2; or IER3, MEST and TGFB2 at a detectably higherlevel than an equivalent number of bone marrow-derived mesenchymal stemcells BM-MSCs, wherein said bone marrow-derived mesenchymal stem cellshave undergone a number of passages in culture equivalent to the numberof passages said isolated placental cells have undergone.

Expression of the above-referenced genes can be assessed by standardtechniques. For example, probes based on the sequence of the gene(s) canbe individually selected and constructed by conventional techniques.Expression of the genes can be assessed, e.g., on a microarraycomprising probes to one or more of the genes, e.g., an AffymetrixGENECHIP® Human Genome U133A 2.0 array, or an Affymetrix GENECHIP® HumanGenome U133 Plus 2.0 (Santa Clara, Calif.). Expression of these genescan be assessed even if the sequence for a particular GenBank accessionnumber is amended because probes specific for the amended sequence canreadily be generated using well-known standard techniques.

The level of expression of these genes can be used to confirm theidentity of a population of isolated placental cells, to identify apopulation of cells as comprising at least a plurality of isolatedplacental cells, or the like. Populations of isolated placental cells,the identity of which is confirmed, can be clonal, e.g., populations ofisolated placental cells expanded from a single isolated placental cell,or a mixed population of stem cells, e.g., a population of cellscomprising solely isolated placental cells that are expanded frommultiple isolated placental cells, or a population of cells comprisingisolated placental cells, as described herein, and at least one othertype of cell.

The level of expression of these genes can be used to select populationsof isolated placental cells. For example, a population of cells, e.g.,clonally-expanded cells, may be selected if the expression of one ormore of the genes listed above is significantly higher in a sample fromthe population of cells than in an equivalent population of mesenchymalstem cells. Such selecting can be of a population from a plurality ofisolated placental cell populations, from a plurality of cellpopulations, the identity of which is not known, etc.

Isolated placental cells can be selected on the basis of the level ofexpression of one or more such genes as compared to the level ofexpression in said one or more genes in, e.g., a mesenchymal stem cellcontrol, for example, the level of expression in said one or more genesin an equivalent number of bone marrow-derived mesenchymal stem cells.In one embodiment, the level of expression of said one or more genes ina sample comprising an equivalent number of mesenchymal stem cells isused as a control. In another embodiment, the control, for isolatedplacental cells tested under certain conditions, is a numeric valuerepresenting the level of expression of said one or more genes inmesenchymal stem cells under said conditions.

The isolated placental cells described herein display the abovecharacteristics (e.g., combinations of cell surface markers and/or geneexpression profiles) in primary culture, or during proliferation inmedium comprising, e.g., DMEM-LG (Gibco), 2% fetal calf serum (FCS)(Hyclone Laboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100U penicillin/1000Ustreptomycin.

In certain embodiments of any of the placental cells disclosed herein,the cells are human. In certain embodiments of any of the placentalcells disclosed herein, the cellular marker characteristics or geneexpression characteristics are human markers or human genes.

In another specific embodiment of said isolated placental cells orpopulations of cells comprising the isolated placental cells, said cellsor population have been expanded, for example, passaged at least, about,or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 times, or proliferated for at least, about, or no morethan, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38 or 40 population doublings.

In another specific embodiment of said isolated placental cells orpopulations of cells comprising the isolated placental cells, said cellsor population are primary isolates. In another specific embodiment ofthe isolated placental cells, or populations of cells comprisingisolated placental cells, that are disclosed herein, said isolatedplacental cells are fetal in origin (that is, have the fetal genotype).

In certain embodiments, said isolated placental cells do notdifferentiate during culturing in growth medium, i.e., medium formulatedto promote proliferation, e.g., during proliferation in growth medium.In another specific embodiment, said isolated placental cells do notrequire a feeder layer in order to proliferate. In another specificembodiment, said isolated placental cells do not differentiate inculture in the absence of a feeder layer, solely because of the lack ofa feeder cell layer.

In another embodiment, cells useful in the methods and compositionsdescribed herein are isolated placental cells, wherein a plurality ofsaid isolated placental cells are positive for aldehyde dehydrogenase(ALDH), as assessed by an aldehyde dehydrogenase activity assay. Suchassays are known in the art (see, e.g., Bostian and Betts, Biochem. J.,173, 787, (1978)). In a specific embodiment, said ALDH assay usesALDEFLUOR® (Aldagen, Inc., Ashland, Oreg.) as a marker of aldehydedehydrogenase activity. In a specific embodiment, said plurality isbetween about 3% and about 25% of cells in said population of cells. Inanother embodiment, provided herein is a population of isolatedumbilical cord cells, e.g., multipotent isolated umbilical cord cells,wherein a plurality of said isolated umbilical cord cells are positivefor aldehyde dehydrogenase, as assessed by an aldehyde dehydrogenaseactivity assay that uses ALDEFLUOR® as an indicator of aldehydedehydrogenase activity. In a specific embodiment, said plurality isbetween about 3% and about 25% of cells in said population of cells. Inanother embodiment, said population of isolated placental cells orisolated umbilical cord cells shows at least three-fold, or at leastfive-fold, higher ALDH activity than a population of bone marrow-derivedmesenchymal stem cells having about the same number of cells andcultured under the same conditions.

In certain embodiments of any of the populations of cells comprising theisolated placental cells described herein, the placental cells in saidpopulations of cells are substantially free of cells having a maternalgenotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%; 75%, 80%,85%, 90%, 95%, 98% or 99% of the placental cells in said population havea fetal genotype. In certain other embodiments of any of the populationsof cells comprising the isolated placental cells described herein, thepopulations of cells comprising said placental cells are substantiallyfree of cells having a maternal genotype; e.g., at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells insaid population have a fetal genotype.

In a specific embodiment of any of the above isolated placental cells orcell populations of isolated placental cells, the karyotype of thecells, or at least about 95% or about 99% of the cells in saidpopulation, is normal. In another specific embodiment of any of theabove placental cells or cell populations, the cells, or cells in thepopulation of cells, are non-maternal in origin.

Isolated placental cells, or populations of isolated placental cells,bearing any of the above combinations of markers, can be combined in anyratio. Any two or more of the above isolated placental cell populationscan be combined to form an isolated placental cell population. Forexample, an population of isolated placental cells can comprise a firstpopulation of isolated placental cells defined by one of the markercombinations described above, and a second population of isolatedplacental cells defined by another of the marker combinations describedabove, wherein said first and second populations are combined in a ratioof about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40:60,50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about99:1. In like fashion, any three, four, five or more of theabove-described isolated placental cells or isolated placental cellspopulations can be combined.

Isolated placental cells useful in the methods and compositionsdescribed herein can be obtained, e.g., by disruption of placentaltissue, with or without enzymatic digestion (see Section 4.3.3) orperfusion (see Section 4.3.4). For example, populations of isolatedplacental cells can be produced according to a method comprisingperfusing a mammalian placenta that has been drained of cord blood andperfused to remove residual blood; perfusing said placenta with aperfusion solution; and collecting said perfusion solution, wherein saidperfusion solution after perfusion comprises a population of placentalcells that comprises isolated placental cells; and isolating a pluralityof said isolated placental cells from said population of cells. In aspecific embodiment, the perfusion solution is passed through both theumbilical vein and umbilical arteries and collected after it exudes fromthe placenta. In another specific embodiment, the perfusion solution ispassed through the umbilical vein and collected from the umbilicalarteries, or passed through the umbilical arteries and collected fromthe umbilical vein.

In various embodiments, the isolated placental cells, contained within apopulation of cells obtained from perfusion of a placenta, are at least50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said populationof placental cells. In another specific embodiment, the isolatedplacental cells collected by perfusion comprise fetal and maternalcells. In another specific embodiment, the isolated placental cellscollected by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% orat least 99.5% fetal cells.

In another specific embodiment, provided herein is a compositioncomprising a population of the isolated placental cells, as describedherein, collected by perfusion, wherein said composition comprises atleast a portion of the perfusion solution used to collect the isolatedplacental cells.

Isolated populations of the isolated placental cells described hereincan be produced by digesting placental tissue with a tissue-disruptingenzyme to obtain a population of placental cells comprising the cells,and isolating, or substantially isolating, a plurality of the placentalcells from the remainder of said placental cells. The whole, or any partof, the placenta can be digested to obtain the isolated placental cellsdescribed herein. In specific embodiments, for example, said placentaltissue can be a whole placenta, an amniotic membrane, chorion, acombination of amnion and chorion, or a combination of any of theforegoing. In other specific embodiment, the tissue-disrupting enzyme istrypsin or collagenase. In various embodiments, the isolated placentalcells, contained within a population of cells obtained from digesting aplacenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least99.5% of said population of placental cells.

The isolated populations of placental cells described above, andpopulations of isolated placental cells generally, can comprise about,at least, or no more than, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×1×10¹¹ or more of the isolatedplacental cells. Populations of isolated placental cells useful in themethods of treatment described herein comprise at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% viable isolated placentalcells, e.g., as determined by, e.g., trypan blue exclusion

4.2.3 Growth in Culture

The growth of the isolated placental cells described herein in Section4.4.2, as for any mammalian cell, depends in part upon the particularmedium selected for growth. Under optimum conditions, the isolatedplacental cells typically double in number in about 1 day. Duringculture, the isolated placental cells described herein adhere to asubstrate in culture, e.g. the surface of a tissue culture container(e.g., tissue culture dish plastic, fibronectin-coated plastic, and thelike) and form a monolayer.

Populations of placental cells that comprise the isolated placentalcells described herein, when cultured under appropriate conditions, canform embryoid-like bodies, that is, three-dimensional clusters of cellsgrow atop the adherent cell layer. Cells within the embryoid-like bodiescan express markers associated with very early stem cells, e.g., OCT-4,Nanog, SSEA3 and SSEA4. Cells within the embryoid-like bodies aretypically not adherent to the culture substrate, as are the isolatedplacental cells described herein, but tend to remain attached to theadherent cells during culture. Embryoid-like body cells are dependentupon the adherent isolated placental cells for viability, asembryoid-like bodies do not form in the absence of the adherent isolatedplacental cells. The adherent isolated placental cells thus facilitatethe growth of one or more embryoid-like bodies in a population ofplacental cells that comprise the adherent isolated placental cells.Without wishing to be bound by theory, the cells of the embryoid-likebodies are thought to grow on the adherent isolated placental cells muchas embryonic stem cells grow on a feeder layer of cells.

4.3 Methods of Obtaining Isolated Placental Cells

4.3.1 Stem Cell Collection Composition

Further provided herein are methods of collecting and isolatingplacental cells, e.g., the isolated placental cells described in Section4.2.2, above. Generally, such cells are obtained from a mammalianplacenta using a physiologically-acceptable solution, e.g., a cellcollection composition. An exemplary cell collection composition isdescribed in detail in related U.S. Patent Application Publication No.2007/0190042, entitled “Improved Medium for Collecting Placental StemCells and Preserving Organs,” the disclosure of which is incorporatedherein by reference in its entirety

The cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of cells, e.g., the isolated placental cells described herein,for example, a saline solution (e.g., phosphate-buffered saline, Kreb'ssolution, modified Kreb's solution, Eagle's solution, 0.9% NaCl. etc.),a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.

The cell collection composition can comprise one or more components thattend to preserve isolated placental cells, that is, prevent the isolatedplacental cells from dying, or delay the death of the isolated placentalcells, reduce the number of isolated placental cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like. In one embodiment, the antibioticis gentamycin, e.g., about 0.005% to about 0.01% (w/v) in culture medium

The cell collection composition can also comprise one or more of thefollowing compounds: adenosine (about 1 mM to about 50 mM); D-glucose(about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50mM); a macromolecule of molecular weight greater than 20,000 daltons, inone embodiment, present in an amount sufficient to maintain endothelialintegrity and cellular viability (e.g., a synthetic or naturallyoccurring colloid, a polysaccharide such as dextran or a polyethyleneglycol present at about 25 g/l to about 100 g/l, or about 40 g/l toabout 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylatedhydroxytoluene, glutathione, vitamin C or vitamin E present at about 25μM to about 100 μM); a reducing agent (e.g., N-acetylcysteine present atabout 0.1 mM to about 5 mM); an agent that prevents calcium entry intocells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

4.3.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the isolatedplacental cells harvested therefrom. For example, isolated humanplacental cells can be used, in light of the medical history, forpersonalized medicine for the infant associated with the placenta, orfor parents, siblings or other relatives of the infant.

Prior to recovery of isolated placental cells, the umbilical cord bloodand placental blood are preferably removed. In certain embodiments,after delivery, the cord blood in the placenta is recovered. Theplacenta can be subjected to a conventional cord blood recovery process.Typically a needle or cannula is used, with the aid of gravity, toexsanguinate the placenta (see, e.g., Anderson, U.S. Pat. No. 5,372,581;Hessel et al., U.S. Pat. No. 5,415,665). The needle or cannula isusually placed in the umbilical vein and the placenta can be gentlymassaged to aid in draining cord blood from the placenta. Such cordblood recovery may be performed commercially, e.g., LifeBank USA, CedarKnolls, N.J. Preferably, the placenta is gravity drained without furthermanipulation so as to minimize tissue disruption during cord bloodrecovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. Pat. No. 7,147,626, the disclosure of which is incorporatedby reference herein. Preferably, the placenta is delivered to thelaboratory four to twenty-four hours following delivery. In certainembodiments, the proximal umbilical cord is clamped, preferably within4-5 cm (centimeter) of the insertion into the placental disc prior tocord blood recovery. In other embodiments, the proximal umbilical cordis clamped after cord blood recovery but prior to further processing ofthe placenta.

The placenta, prior to cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5° C.to 25° C. The placenta may be stored for a period of for a period offour to twenty-four hours, up to forty-eight hours, or longer than fortyeight hours, prior to perfusing the placenta to remove any residual cordblood. In one embodiment, the placenta is harvested from between aboutzero hours to about two hours post-expulsion. The placenta is preferablystored in an anticoagulant solution at a temperature of 5° C. to 25° C.Suitable anticoagulant solutions are well known in the art. For example,a solution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta ispreferably stored for no more than 36 hours before placental cells arecollected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain isolated placental cells.

4.3.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, stem cells are collected from a mammalian placenta byphysical disruption of part of all of the organ. For example, theplacenta, or a portion thereof, may be, e.g., crushed, sheared, minced,diced, chopped, macerated or the like. The tissue can then be culturedto obtain a population of isolated placental cells. Typically, theplacental tissue is disrupted using, e.g., culture medium, a salinesolution, or a stem cell collection composition (see Section 4.5.1 andbelow).

The placenta can be dissected into components prior to physicaldisruption and/or enzymatic digestion and stem cell recovery. Isolatedplacental cells can be obtained from all or a portion of the amnioticmembrane, chorion, umbilical cord, placental cotyledons, or anycombination thereof, including from a whole placenta. Preferably,isolated placental cells are obtained from placental tissue comprisingamnion and chorion. Typically, isolated placental cells can be obtainedby disruption of a small block of placental tissue, e.g., a block ofplacental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 orabout 1000 cubic millimeters in volume. Any method of physicaldisruption can be used, provided that the method of disruption leaves aplurality, more preferably a majority, and more preferably at least 60%,70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable, asdetermined by, e.g., trypan blue exclusion.

The isolated adherent placental cells can generally be collected from aplacenta, or portion thereof, at any time within about the first threedays post-expulsion, but preferably between about 8 hours and about 18hours post-expulsion.

In a specific embodiment, the disrupted tissue is cultured in tissueculture medium suitable for the proliferation of isolated placentalcells (see, e.g., Section 4.6, below, describing the culture ofplacental cells, e.g., PDACs).

In another specific embodiment, isolated placental cells are collectedby physical disruption of placental tissue, wherein the physicaldisruption includes enzymatic digestion, which can be accomplished byuse of one or more tissue-digesting enzymes. The placenta, or a portionthereof, may also be physically disrupted and digested with one or moreenzymes, and the resulting material then immersed in, or mixed into, acell collection composition.

A preferred cell collection composition comprises one or moretissue-disruptive enzyme(s). Enzymes that can be used to disruptplacenta tissue include papain, deoxyribonucleases, serine proteases,such as trypsin, chymotrypsin, collagenase, dispase or elastase. Serineproteases may be inhibited by alpha 2 microglobulin in serum andtherefore the medium used for digestion is usually serum-free. EDTA andDNase are commonly used in enzyme digestion procedures to increase theefficiency of cell recovery. The digestate is preferably diluted so asto avoid trapping cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for digestion using trypsin include, 0.1% to about 2%trypsin, e.g., about 0.25% trypsin. Proteases can be used incombination, that is, two or more proteases in the same digestionreaction, or can be used sequentially in order to liberate placentalcells, e.g., placental stem cells and placental multipotent cells. Forexample, in one embodiment, a placenta, or part thereof, is digestedfirst with an appropriate amount of collagenase I at about 1 to about 2mg/ml for, e.g., 30 minutes, followed by digestion with trypsin, at aconcentration of about 0.25%, for, e.g., 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid(EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the stem cells with the stem cell collectioncomposition.

Following digestion, the digestate is washed, for example, three timeswith culture medium, and the washed cells are seeded into cultureflasks. The cells are then isolated by differential adherence, andcharacterized for, e.g., viability, cell surface markers,differentiation, and the like.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental cells isolated can comprise a mix of placental cells derivedfrom both fetal and maternal sources. Where a portion of the placentathat comprises no, or a negligible number of, maternal cells (forexample, amnion), the placental cells isolated therefrom will comprisealmost exclusively fetal placental cells (that is, placental cellshaving the genotype of the fetus).

Placental cells, e.g., the placental cells described in Section 4.2.2,above, can be isolated from disrupted placental tissue by differentialtrypsinization (see Section 4.3.5, below) followed by culture in one ormore new culture containers in fresh proliferation medium, optionallyfollowed by a second differential trypsinization step.

4.3.4 Placental Perfusion

Placental cells, e.g., the placental cells described in Section 4.2.2,above, can also be obtained by perfusion of the mammalian placenta.Methods of perfusing mammalian placenta to obtain placental cells aredisclosed, e.g., in Hariri, U.S. Pat. Nos. 7,045,148 and 7,255,729, inU.S. Patent Application Publication Nos. 2007/0275362 and 2007/0190042,the disclosures of each of which are incorporated herein by reference intheir entireties.

Placental cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a cell collection composition as aperfusion solution. In one embodiment, a mammalian placenta is perfusedby passage of perfusion solution through either or both of the umbilicalartery and umbilical vein. The flow of perfusion solution through theplacenta may be accomplished using, e.g., gravity flow into theplacenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid through the placentalvasculature and surrounding tissue. The placenta can also be perfused bypassage of a perfusion fluid into the umbilical vein and collection fromthe umbilical arteries, or passage of a perfusion fluid into theumbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilicalvein are connected simultaneously, e.g., to a pipette that is connectedvia a flexible connector to a reservoir of the perfusion solution. Theperfusion solution is passed into the umbilical vein and artery. Theperfusion solution exudes from and/or passes through the walls of theblood vessels into the surrounding tissues of the placenta, and iscollected in a suitable open vessel from the surface of the placentathat was attached to the uterus of the mother during gestation. Theperfusion solution may also be introduced through the umbilical cordopening and allowed to flow or percolate out of openings in the wall ofthe placenta which interfaced with the maternal uterine wall. Placentalcells that are collected by this method, which can be referred to as a“pan” method, are typically a mixture of fetal and maternal cells.

In another embodiment, the perfusion solution is passed through theumbilical veins and collected from the umbilical artery, or is passedthrough the umbilical artery and collected from the umbilical veins.Placental cells collected by this method, which can be referred to as a“closed circuit” method, are typically almost exclusively fetal.

It will be appreciated that perfusion using the pan method, that is,whereby perfusate is collected after it has exuded from the maternalside of the placenta, results in a mix of fetal and maternal cells. As aresult, the cells collected by this method can comprise a mixedpopulation of placental cells, e.g., placental stem cells or placentalmultipotent cells, of both fetal and maternal origin. In contrast,perfusion solely through the placental vasculature in the closed circuitmethod, whereby perfusion fluid is passed through one or two placentalvessels and is collected solely through the remaining vessel(s), resultsin the collection of a population of placental cells almost exclusivelyof fetal origin.

The closed circuit perfusion method can, in one embodiment, be performedas follows. A post-partum placenta is obtained within about 48 hoursafter birth. The umbilical cord is clamped and cut above the clamp. Theumbilical cord can be discarded, or can processed to recover, e.g.,umbilical cord stem cells, and/or to process the umbilical cord membranefor the production of a biomaterial. The amniotic membrane can beretained during perfusion, or can be separated from the chorion, e.g.,using blunt dissection with the fingers. If the amniotic membrane isseparated from the chorion prior to perfusion, it can be, e.g.,discarded, or processed, e.g., to obtain stem cells by enzymaticdigestion, or to produce, e.g., an amniotic membrane biomaterial, e.g.,the biomaterial described in U.S. Application Publication No.2004/0048796, the disclosure of which is incorporated by referenceherein in its entirety. After cleaning the placenta of all visible bloodclots and residual blood, e.g., using sterile gauze, the umbilical cordvessels are exposed, e.g., by partially cutting the umbilical cordmembrane to expose a cross-section of the cord. The vessels areidentified, and opened, e.g., by advancing a closed alligator clampthrough the cut end of each vessel. The apparatus, e.g., plastic tubingconnected to a perfusion device or peristaltic pump, is then insertedinto each of the placental arteries. The pump can be any pump suitablefor the purpose, e.g., a peristaltic pump. Plastic tubing, connected toa sterile collection reservoir, e.g., a blood bag such as a 250 mLcollection bag, is then inserted into the placental vein. Alternatively,the tubing connected to the pump is inserted into the placental vein,and tubes to a collection reservoir(s) are inserted into one or both ofthe placental arteries. The placenta is then perfused with a volume ofperfusion solution, e.g., about 750 ml of perfusion solution. Cells inthe perfusate are then collected, e.g., by centrifugation. In certainembodiments, the placenta is perfused with perfusion solution, e.g.,100-300 mL perfusion solution, to remove residual blood prior toperfusion to collect placental cells, e.g., placental stem cells and/orplacental multipotent cells. In another embodiment, the placenta is notperfused with perfusion solution to remove residual blood prior toperfusion to collect placental cells.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to isolate placental cells may varydepending upon the number of cells to be collected, the size of theplacenta, the number of collections to be made from a single placenta,etc. In various embodiments, the volume of perfusion liquid may be from50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL,250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically,the placenta is perfused with 700-800 mL of perfusion liquid followingexsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe cell collection composition, or a standard perfusion solution (e.g.,a normal saline solution such as phosphate buffered saline (“PBS”)) withor without an anticoagulant (e.g., heparin, warfarin sodium, coumarin,bishydroxycoumarin), and/or with or without an antimicrobial agent(e.g., β-mercaptoethanol (0.1 mM); antibiotics such as streptomycin(e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml), amphotericin B(e.g., at 0.5 μg/ml). In one embodiment, an isolated placenta ismaintained or cultured for a period of time without collecting theperfusate, such that the placenta is maintained or cultured for 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours, or 2 or 3 or more days before perfusion and collectionof perfusate. The perfused placenta can be maintained for one or moreadditional time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused asecond time with, e.g., 700-800 mL perfusion fluid. The placenta can beperfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3,4, 5 or 6 hours. In a preferred embodiment, perfusion of the placentaand collection of perfusion solution, e.g., cell collection composition,is repeated until the number of recovered nucleated cells falls below100 cells/ml. The perfusates at different time points can be furtherprocessed individually to recover time-dependent populations of cells,e.g., stem cells. Perfusates from different time points can also bepooled. In a preferred embodiment, placental cells are collected at atime or times between about 8 hours and about 18 hours post-expulsion.

Perfusion preferably results in the collection of significantly moreplacental cells than the number obtainable from a mammalian placenta notperfused with said solution, and not otherwise treated to obtainplacental cells (e.g., by tissue disruption, e.g., enzymatic digestion).In this context, “significantly more” means at least 10% more. Perfusionyields significantly more placental cells than, e.g., the number ofplacental cells isolatable from culture medium in which a placenta, orportion thereof, has been cultured.

Placental cells can be isolated from placenta by perfusion with asolution comprising one or more proteases or other tissue-disruptiveenzymes. In a specific embodiment, a placenta or portion thereof (e.g.,amniotic membrane, amnion and chorion, placental lobule or cotyledon,umbilical cord, or combination of any of the foregoing) is brought to25-37° C., and is incubated with one or more tissue-disruptive enzymesin 200 mL of a culture medium for 30 minutes. Cells from the perfusateare collected, brought to 4° C., and washed with a cold inhibitor mixcomprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol.The placental cells are washed after several minutes with a cold (e.g.,4° C.) stem cell collection composition.

4.3.5 Isolation, Sorting, and Characterization of Placental Cells

The isolated placental cells, e.g., the cells described in Section4.2.2, above, whether obtained by perfusion or physical disruption,e.g., by enzymatic digestion, can initially be purified from (i.e., beisolated from) other cells by Ficoll gradient centrifugation. Suchcentrifugation can follow any standard protocol for centrifugationspeed, etc. In one embodiment, for example, cells collected from theplacenta are recovered from perfusate by centrifugation at 5000×g for 15minutes at room temperature, which separates cells from, e.g.,contaminating debris and platelets. In another embodiment, placentalperfusate is concentrated to about 200 ml, gently layered over Ficoll,and centrifuged at about 1100×g for 20 minutes at 22° C., and thelow-density interface layer of cells is collected for furtherprocessing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for cell maintenance, e.g., stem cellmaintenance, for example, IMDM serum-free medium containing 2 U/mlheparin and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cellfraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo,Norway) according to the manufacturer's recommended procedure.

Placental cells obtained by perfusion or digestion can, for example, befurther, or initially, isolated by differential trypsinization using,e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because the isolated placentalcells, which are tissue culture plastic-adherent, typically detach fromthe plastic surfaces within about five minutes whereas other adherentpopulations typically require more than 20-30 minutes incubation. Thedetached placental cells can be harvested following trypsinization andtrypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS,Cambrex). In one embodiment of isolation of adherent cells, aliquots of,for example, about 5−10×10⁶ cells are placed in each of several T-75flasks, preferably fibronectin-coated T75 flasks. In such an embodiment,the cells can be cultured with commercially available Mesenchymal StemCell Growth Medium (MSCGM) (Cambrex), and placed in a tissue cultureincubator (37° C., 5% CO₂). After 10 to 15 days, non-adherent cells areremoved from the flasks by washing with PBS. The PBS is then replaced byMSCGM. Flasks are preferably examined daily for the presence of variousadherent cell types and in particular, for identification and expansionof clusters of fibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34; if so, the cell is CD34⁺. Likewise, if a cellproduces enough OCT-4 RNA to be detectable by RT-PCR, or significantlymore OCT-4 RNA than an adult cell, the cell is OCT-4⁺. Antibodies tocell surface markers (e.g., CD markers such as CD34) and the sequence ofstem cell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laserexcitation of fluorescent moieties in the individual particles resultsin a small electrical charge allowing electromagnetic separation ofpositive and negative particles from a mixture. In one embodiment, cellsurface marker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, cells from placenta, e.g., PDACs are sorted onthe basis of expression of one or more of the markers CD34, CD38, CD44,CD45, CD73, CD105, OCT-4 and/or HLA-G. This can be accomplished inconnection with procedures to select such cells on the basis of theiradherence properties in culture. For example, tissue culture plasticadherence selection can be accomplished before or after sorting on thebasis of marker expression. In one embodiment, for example, cells aresorted first on the basis of their expression of CD34; CD34⁻ cells areretained, and CD34⁻ cells that are additionally CD200⁺ and HLA-G⁻ areseparated from all other CD34⁻ cells. In another embodiment, cells fromplacenta are sorted based on their expression of markers CD200 and/orHLA-G; for example, cells displaying CD200 and lacking HLA-G areisolated for further use. Cells that express, e.g., CD200 and/or lack,e.g., HLA-G can, in a specific embodiment, be further sorted based ontheir expression of CD73 and/or CD105, or epitopes recognized byantibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45.For example, in another embodiment, placental cells are sorted byexpression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38and CD45, and placental cells that are CD200⁺, HLA-G⁻, CD73⁺, CD105⁺,CD34⁻, CD38⁻ and CD45⁻ are isolated from other placental cells forfurther use.

In specific embodiments of any of the above embodiments of sortedplacental cells, at least 50%, 60%, 70%, 80%, 90% or 95% of the cells ina cell population remaining after sorting are said isolated placentalcells. Placental cells can be sorted by one or more of any of themarkers described in Section 4.2.2, above.

In a specific embodiment, for example, placental cells that are (1)adherent to tissue culture plastic, and (2) CD10⁺, CD34⁻ and CD105⁺ aresorted from (i.e., isolated from) other placental cells. In anotherspecific embodiment, placental cells that are (1) adherent to tissueculture plastic, and (2) CD10⁺, CD34⁻, CD105⁺ and CD200⁺ are sorted from(i.e., isolated from) other placental cells. In another specificembodiment, placental cells that are (1) adherent to tissue cultureplastic, and (2) CD10⁺, CD34⁻, CD45⁻, CD90⁺, CD105⁺ and CD200⁺ aresorted from (i.e., isolated from) other placental cells.

With respect to nucleotide sequence-based detection of placental cells,sequences for the markers listed herein are readily available inpublicly-available databases such as GenBank or EMBL.

With respect to antibody-mediated detection and sorting of placentalcells, e.g., placental stem cells or placental multipotent cells, anyantibody, specific for a particular marker, can be used, in combinationwith any fluorophore or other label suitable for the detection andsorting of cells (e.g., fluorescence-activated cell sorting).Antibody/fluorophore combinations to specific markers include, but arenot limited to, fluorescein isothiocyanate (FITC) conjugated monoclonalantibodies against HLA-G (available from Serotec, Raleigh, N.C.), CD10(available from BD Immunocytometry Systems, San Jose, Calif.), CD44(available from BD Biosciences Pharmingen, San Jose, Calif.), and CD105(available from R&D Systems Inc., Minneapolis, Minn.); phycoerythrin(PE) conjugated monoclonal antibodies against CD44, CD200, CD117, andCD13 (BD Biosciences Pharmingen); phycoerythrin-Cy7 (PE Cy7) conjugatedmonoclonal antibodies against CD33 and CD10 (BD Biosciences Pharmingen);allophycocyanin (APC) conjugated streptavidin and monoclonal antibodiesagainst CD38 (BD Biosciences Pharmingen); and Biotinylated CD90 (BDBiosciences Pharmingen). Other antibodies that can be used include, butare not limited to, CD133-APC (Miltenyi), KDR-Biotin (CD309, Abcam),CytokeratinK-Fitc (Sigma or Dako), HLA ABC-Fitc (BD), HLA DR,DQ,DP-PE(BD), β-2-microglobulin-PE (BD), CD80-PE (BD) and CD86-APC (BD). Otherantibody/label combinations that can be used include, but are notlimited to, CD45-PerCP (peridin chlorophyllprotein); CD44-PE; CD19-PE;CD10-F (fluorescein); HLA-G-F and 7-amino-actinomycin-D (7-AAD);HLA-ABC-F; and the like. This list is not exhaustive, and otherantibodies from other suppliers are also commercially available.

The isolated placental cells provided herein can be assayed for CD117 orCD133 using, for example, phycoerythrin-Cy5 (PE Cy5) conjugatedstreptavidin and biotin conjugated monoclonal antibodies against CD117or CD133; however, using this system, the cells can appear to bepositive for CD117 or CD133, respectively, because of a relatively highbackground.

The isolated placental cells can be labeled with an antibody to a singlemarker and detected and/sorted. Placental cells can also besimultaneously labeled with multiple antibodies to different markers.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Isolated placental cells can also be characterized and/or sorted basedon cell morphology and growth characteristics. For example, isolatedplacental cells can be characterized as having, and/or selected on thebasis of, e.g., a fibroblastoid appearance in culture. The isolatedplacental cells can also be characterized as having, and/or be selected,on the basis of their ability to form embryoid-like bodies. In oneembodiment, for example, placental cells that are fibroblastoid inshape, express CD73 and CD105, and produce one or more embryoid-likebodies in culture are isolated from other placental cells. In anotherembodiment, OCT-4⁺ placental cells that produce one or moreembryoid-like bodies in culture are isolated from other placental cells.

In another embodiment, isolated placental cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as MESENCULT™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia).

The isolated placental cells can be assessed for viability,proliferation potential, and longevity using standard techniques knownin the art, such as trypan blue exclusion assay, fluorescein diacetateuptake assay, propidium iodide uptake assay (to assess viability); andthymidine uptake assay, MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellproliferation assay (to assess proliferation). Longevity may bedetermined by methods well known in the art, such as by determining themaximum number of population doubling in an extended culture.

Isolated placental cells, e.g., the isolated placental cells describedin Section 4.2.2, above, can also be separated from other placentalcells using other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

4.4 Culture of Isolated Placental Cells

4.4.1 Culture Media

Isolated placental cells, or populations of isolated placental cells, orcells or placental tissue from which placental cells grow out, can beused to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (e.g.,native or denatured), gelatin, fibronectin, ornithine, vitronectin, andextracellular membrane protein (e.g., MATRIGEL® (BD Discovery Labware,Bedford, Mass.)).

Isolated placental cells can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture ofcells, e.g., stem cells. Preferably, the culture medium comprises serum.The isolated placental cells can be cultured in, for example, DMEM-LG(Dulbecco's Modified Essential Medium, low glucose)/MCDB 201 (chickfibroblast basal medium) containing ITS (insulin-transferrin-selenium),LA+BSA (linoleic acid-bovine serum albumin), dexamethasone L-ascorbicacid, PDGF, EGF, IGF-1, and penicillin/streptomycin; DMEM-HG (highglucose) comprising 10% fetal bovine serum (FBS); DMEM-HG comprising 15%FBS; IMDM (Iscove's modified Dulbecco's medium) comprising 10% FBS, 10%horse serum, and hydrocortisone; M199 comprising 1% to 20% FBS, EGF, andheparin; α-MEM (minimal essential medium) comprising 10% FBS, GLUTAMAX™and gentamicin; DMEM comprising 10% FBS, GLUTAMAX™ and gentamicin, etc.

Other media in that can be used to culture placental cells include DMEM(high or low glucose), Eagle's basal medium, Ham's F10 medium (F10),Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

The isolated placental cells can be cultured in standard tissue cultureconditions, e.g., in tissue culture dishes or multiwell plates. Theisolated placental cells can also be cultured using a hanging dropmethod. In this method, isolated placental cells are suspended at about1×10⁴ cells per mL in about 5 mL of medium, and one or more drops of themedium are placed on the inside of the lid of a tissue culturecontainer, e.g., a 100 mL Petri dish. The drops can be, e.g., singledrops, or multiple drops from, e.g., a multichannel pipetter. The lid iscarefully inverted and placed on top of the bottom of the dish, whichcontains a volume of liquid, e.g., sterile PBS sufficient to maintainthe moisture content in the dish atmosphere, and the stem cells arecultured.

In one embodiment, isolated placental cells are cultured in the presenceof a compound that acts to maintain an undifferentiated phenotype in theisolated placental cells. In a specific embodiment, the compound is asubstituted 3,4-dihydropyridimol[4,5-d]pyrimidine. In another specificembodiment, the compound is a compound having the following chemicalstructure:

The compound can be contacted with isolated placental cells, or apopulation of isolated placental cells, at a concentration of, forexample, between about 1 μM to about 10 μM.

4.4.2 Expansion and Proliferation of Placental Cells

Once an isolated placental cell, or population of isolated placentalcells (e.g., a placental cell or population of placental cells separatedfrom at least 50% of the placental cells with which the stem cell orpopulation of stem cells is normally associated in vivo), the cell orpopulation of cells can be proliferated and expanded in vitro. Forexample, a population of the isolated placental cells can be cultured intissue culture containers, e.g., dishes, flasks, multiwell plates, orthe like, for a sufficient time for the cells to proliferate to 70-90%confluence, that is, until the cells and their progeny occupy 70-90% ofthe culturing surface area of the tissue culture container.

The isolated placental cells can be seeded in culture vessels at adensity that allows cell growth. For example, the cells may be seeded atlow density (e.g., about 1,000 to about 5,000 cells/cm²) to high density(e.g., about 50,000 or more cells/cm²). In a preferred embodiment, thecells are cultured in the presence of about 0 to about 5 percent byvolume CO₂ in air. In some preferred embodiments, the cells are culturedat about 2 to about 25 percent O₂ in air, preferably about 5 to about 20percent O₂ in air. The cells preferably are cultured at about 25° C. toabout 40° C., preferably 37° C. The cells are preferably cultured in anincubator. The culture medium can be static or agitated, for example,using a bioreactor. Placental cells, e.g., placental stem cells orplacental multipotent cells, preferably are grown under low oxidativestress (e.g., with addition of glutathione, ascorbic acid, catalase,tocopherol, N-acetylcysteine, or the like).

Once confluence of less than 100%, for example, 70% to 90% is obtained,the cells may be passaged. For example, the cells can be enzymaticallytreated, e.g., trypsinized, using techniques well-known in the art, toseparate them from the tissue culture surface. After removing the cellsby pipetting and counting the cells, about 10,000-100,000 cells/cm² arepassaged to a new culture container containing fresh culture medium.Typically, the new medium is the same type of medium from which theisolated placental cells were removed. The isolated placental cells canbe passaged about, at least, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 times, or more.

4.4.3 Populations of Isolated Placental Cells

Also provided herein are populations of isolated placental cells, e.g.,the isolated placental cells described in Section 4.2.2, above, usefulin the methods and compositions described herein. Populations ofisolated placental cells can be isolated directly from one or moreplacentas; that is, the cell population can be a population of placentalcells comprising the isolated placental cells, wherein the isolatedplacental cells are obtained from, or contained within, perfusate, orobtained from, or contained within, disrupted placental tissue, e.g.,placental tissue digestate (that is, the collection of cells obtained byenzymatic digestion of a placenta or part thereof). The isolatedplacental cells described herein can also be cultured and expanded toproduce populations of the isolated placental cells. Populations ofplacental cells comprising the isolated placental cells can also becultured and expanded to produce placental cell populations.

Placental cell populations useful in the methods of treatment providedherein comprise the isolated placental cells, for example, the isolatedplacental cells as described in Section 4.4.2 herein. In variousembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 99% of the cells in a placental cell population are the isolatedplacental cells. That is, a population of the isolated placental cellscan comprise, e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% cells that are not the isolated placental cells.

Isolated placental cell populations useful in the methods andcompositions described herein can be produced by, e.g., selectingisolated placental cells, whether derived from enzymatic digestion orperfusion, that express particular markers and/or particular culture ormorphological characteristics. In one embodiment, for example, providedherein is a method of producing a cell population by selecting placentalcells that (a) adhere to a substrate, and (b) express CD200 and lackexpression of HLA-G; and isolating said cells from other cells to form acell population. In another embodiment, a cell population is produced byselecting placental cells that express CD200 and lack expression ofHLA-G, and isolating said cells from other cells to form a cellpopulation. In another embodiment, a cell population is produced byselecting placental cells that (a) adhere to a substrate, and (b)express CD73, CD105, and CD200; and isolating said cells from othercells to form a cell population. In another embodiment, a cellpopulation is produced by identifying placental cells that express CD73,CD105, and CD200, and isolating said cells from other cells to form acell population. In another embodiment, a cell population is produced byselecting placental cells that (a) adhere to a substrate and (b) expressCD200 and OCT-4; and isolating said cells from other cells to form acell population. In another embodiment, a cell population is produced byselecting placental cells that express CD200 and OCT-4, and isolatingsaid cells from other cells to form a cell population. In anotherembodiment, a cell population is produced by selecting placental cellsthat (a) adhere to a substrate, (b) express CD73 and CD105, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In another embodiment, a cell population is producedby selecting placental cells that express CD73 and CD105, and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow for the formation of anembryoid-like body, and isolating said cells from other cells to form acell population. In another embodiment, a cell population is produced byselecting placental cells that (a) adhere to a substrate, and (b)express CD73 and CD105, and lack expression of HLA-G; and isolating saidcells from other cells to form a cell population. In another embodiment,a cell population is produced by selecting placental cells that expressCD73 and CD105 and lack expression of HLA-G, and isolating said cellsfrom other cells to form a cell population. In another embodiment, themethod of producing a cell population comprises selecting placentalcells that (a) adhere to a substrate, (b) express OCT-4, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In another embodiment, a cell population is producedby selecting placental cells that express OCT-4, and facilitate theformation of one or more embryoid-like bodies in a population ofplacental cells comprising said stem cell when said population iscultured under conditions that allow for the formation of anembryoid-like body, and isolating said cells from other cells to form acell population.

In another embodiment, a cell population is produced by selectingplacental cells that (a) adhere to a substrate, and (b) express CD10 andCD105, and do not express CD34; and isolating said cells from othercells to form a cell population. In another embodiment, a cellpopulation is produced by selecting placental cells that express CD10and CD105, and do not express CD34, and isolating said cells from othercells to form a cell population. In another embodiment, a cellpopulation is produced by selecting placental cells that (a) adhere to asubstrate, and (b) express CD10, CD105, and CD200, and do not expressCD34; and isolating said cells from other cells to form a cellpopulation. In another embodiment, a cell population is produced byselecting placental cells that express CD10, CD105, and CD200, and donot express CD34, and isolating said cells from other cells to form acell population. In another specific embodiment, a cell population isproduced by selecting placental cells that (a) adhere to a substrate,and (b) express CD10, CD90, CD105 and CD200, and do not express CD34 andCD45; and isolating said cells from other cells to form a cellpopulation. In another specific embodiment, a cell population isproduced by selecting placental cells that express CD10, CD90, CD105 andCD200, and do not express CD34 and CD45, and isolating said cells fromother cells to form a cell population.

Selection of cell populations comprising placental cells having any ofthe marker combinations described in Section 4.2.2, above, can beisolated or obtained in similar fashion.

In any of the above embodiments, selection of the isolated cellpopulations can additionally comprise selecting placental cells thatexpress ABC-p (a placenta-specific ABC transporter protein; see, e.g.,Allikmets et al., Cancer Res. 58(23):5337-9 (1998)). The method can alsocomprise selecting cells exhibiting at least one characteristic specificto, e.g., a mesenchymal stem cell, for example, expression of CD44,expression of CD90, or expression of a combination of the foregoing.

In the above embodiments, the substrate can be any surface on whichculture and/or selection of cells, e.g., isolated placental cells, canbe accomplished. Typically, the substrate is plastic, e.g., tissueculture dish or multiwell plate plastic. Tissue culture plastic can becoated with a biomolecule, e.g., laminin or fibronectin.

Cells, e.g., isolated placental cells, can be selected for a placentalcell population by any means known in the art of cell selection. Forexample, cells can be selected using an antibody or antibodies to one ormore cell surface markers, for example, in flow cytometry or FACS.Selection can be accomplished using antibodies in conjunction withmagnetic beads. Antibodies that are specific for certain stemcell-related markers are known in the art. For example, antibodies toOCT-4 (Abeam, Cambridge, Mass.), CD200 (Abeam), HLA-G (Abeam), CD73 (BDBiosciences Pharmingen, San Diego, Calif.), CD105 (Abeam; BioDesignInternational, Saco, Me.), etc. Antibodies to other markers are alsoavailable commercially, e.g., CD34, CD38 and CD45 are available from,e.g., StemCell Technologies or BioDesign International.

The isolated placental cell populations can comprise placental cellsthat are not stem cells, or cells that are not placental cells.

The isolated placental cell populations provided herein can be combinedwith one or more populations of non-stem cells or non-placental cells.For example, a population of isolated placental cells can be combinedwith blood (e.g., placental blood or umbilical cord blood),blood-derived stem cells (e.g., stem cells derived from placental bloodor umbilical cord blood), umbilical cord stem cells, populations ofblood-derived nucleated cells, bone marrow-derived mesenchymal cells,bone-derived stem cell populations, crude bone marrow, adult (somatic)stem cells, populations of stem cells contained within tissue, culturedstem cells, populations of fully-differentiated cells (e.g.,chondrocytes, fibroblasts, amniotic cells, osteoblasts, muscle cells,cardiac cells, etc.) and the like. In a specific embodiment, apopulation of cells useful in the methods and compositions describedherein comprises isolated placental cells and isolated umbilical cordcells. Cells in an isolated placental cell population can be combinedwith a plurality of cells of another type in ratios of about100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1,2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1,20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1,50:1, 20:1, 10:1, 5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500;1:1,000; 1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000;1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000;1:20,000,000; 1:50,000,000; or about 1:100,000,000, comparing numbers oftotal nucleated cells in each population. Cells in an isolated placentalcell population can be combined with a plurality of cells of a pluralityof cell types, as well.

In one embodiment, an isolated population of placental cells is combinedwith a plurality of hematopoietic stem cells. Such hematopoietic stemcells can be, for example, contained within unprocessed placental,umbilical cord blood or peripheral blood; in total nucleated cells fromplacental blood, umbilical cord blood or peripheral blood; in anisolated population of CD34⁺ cells from placental blood, umbilical cordblood or peripheral blood; in unprocessed bone marrow; in totalnucleated cells from bone marrow; in an isolated population of CD34⁺cells from bone marrow, or the like.

In other embodiments, a population of the placental cells describedherein, e.g., the PDACs described in Section 4.2.2, above, are combinedwith osteogenic placental adherent cells (OPACs), e.g., the OPACsdescribed in patent application Ser. No. 12/546,556, filed Aug. 24,2009, entitled “Methods and Compositions for Treatment of Bone DefectsWith Placental Stem Cells,” the disclosure of which is herebyincorporated by reference in its entirety.

4.5 Production of a Placental Cell Bank

Isolated cells from postpartum placentas, e.g., the isolated placentalcells described in Section 4.2.2, above, can be cultured in a number ofdifferent ways to produce a set of lots, e.g., wherein a lot is a set ofindividually-administrable doses, of isolated placental cells. Such lotscan, for example, be obtained from cells from placental perfusate orfrom cells from enzyme-digested placental tissue. Sets of lots ofplacental cells, obtained from a plurality of placentas, can be arrangedin a bank of isolated placental cells for, e.g., long-term storage.Generally, tissue culture plastic-adherent placental cells are obtainedfrom an initial culture of placental material to form a seed culture,which is expanded under controlled conditions to form populations ofcells from approximately equivalent numbers of doublings. Lots arepreferably derived from the tissue of a single placenta, but can bederived from the tissue of a plurality of placentas.

In one embodiment, placental cell lots are obtained as follows.Placental tissue is first disrupted, e.g., by mincing, digested with asuitable enzyme, e.g., trypsin or collagenase (see Section 4.3.3,above). The placental tissue preferably comprises, e.g., the entireamnion, entire chorion, or both, from a single placenta, but cancomprise only a part of either the amnion or chorion. The digestedtissue is cultured, e.g., for about 1-3 weeks, preferably about 2 weeks.After removal of non-adherent cells, high-density colonies that form arecollected, e.g., by trypsinization. These cells are collected andresuspended in a convenient volume of culture medium, and are then usedto seed expansion cultures. Expansion cultures can be any arrangement ofseparate cell culture apparatuses, e.g., a Cell Factory by NUNC™. Cellscan be subdivided to any degree so as to seed expansion cultures with,e.g., 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, or10×10⁴ cells/cm². Preferably, from about 1×10³ to about 1×10⁴ cells/cm²are used to seed each expansion culture. The number of expansioncultures may be greater or fewer in number depending upon the particularplacenta(s) from which the cells are obtained.

Expansion cultures are grown until the density of cells in culturereaches a certain value, e.g., about 1×10⁵ cells/cm². Cells can eitherbe collected and cryopreserved at this point, or passaged into newexpansion cultures as described above. Cells can be passaged, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 timesprior to use. A record of the cumulative number of population doublingsis preferably maintained during expansion culture(s). The cells from aculture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, or up to 60doublings. Preferably, however, the number of population doublings,prior to dividing the population of cells into individual doses, is fromabout 15 to about 30. The cells can be culture continuously throughoutthe expansion process, or can be frozen at one or more points duringexpansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 50 million cells per ml, and can comprise between about 10⁶ andabout 10¹⁰ cells in total.

In one embodiment, therefore, a placental cell bank can be made by amethod comprising: expanding primary culture placental cells from ahuman post-partum placenta for a first plurality of populationdoublings; cryopreserving said placental cells to form a Master CellBank; expanding a plurality of placental cells from the Master Cell Bankfor a second plurality of population doublings; cryopreserving saidplacental cells to form a Working Cell Bank; expanding a plurality ofplacental cells from the Working Cell Bank for a third plurality ofpopulation doublings; and cryopreserving said placental cells inindividual doses, wherein said individual doses collectively compose aplacental cell bank. Optionally, a plurality of placental cells fromsaid third plurality of population doublings can be expanded for afourth plurality of population doublings and cryopreserved in individualdoses, wherein said individual doses collectively compose a placentalcell bank.

In another specific embodiment, said primary culture placental cellscomprise placental cells from placental perfusate. In another specificembodiment, said primary culture placental cells comprise placentalcells from digested placental tissue. In another specific embodiment,said primary culture placental cells comprise placental cells fromplacental perfusate and from digested placental tissue. In anotherspecific embodiment, all of said placental cells in said placental cellprimary culture are from the same placenta. In another specificembodiment, the method further comprises the step of selecting CD200⁺ orHLA-G⁻ placental cells from said plurality of said placental cells fromsaid Working Cell Bank to form individual doses. In another specificembodiment, said individual doses comprise from about 10⁴ to about 10⁵placental cells. In another specific embodiment, said individual dosescomprise from about 10⁵ to about 10⁶ placental cells. In anotherspecific embodiment, said individual doses comprise from about 10⁶ toabout 10⁷ placental cells. In another specific embodiment, saidindividual doses comprise from about 10⁷ to about 10⁸ placental cells.In another specific embodiment, said individual doses comprise fromabout 10⁸ to about 10⁹ placental cells. In another specific embodiment,said individual doses comprise from about 10⁹ to about 10¹⁰ placentalcells.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. If the mothertests positive for a tested pathogen, the entire lot from the placentais discarded. Such testing can be performed at any time duringproduction of placental cell lots, e.g., during expansion culture.Pathogens for which the presence is tested can include, withoutlimitation, hepatitis A, hepatitis B, hepatitis C, hepatitis D,hepatitis E, human immunodeficiency virus (types I and II),cytomegalovirus, herpesvirus, and the like.

4.6 Preservation of Placental Cells

Isolated placental cells, e.g., the isolated placental cells describedin Section 4.3.2, above, can be preserved, that is, placed underconditions that allow for long-term storage, or conditions that inhibitcell death by, e.g., apoptosis or necrosis.

Placental cells can be preserved using, e.g., a composition comprisingan apoptosis inhibitor, necrosis inhibitor and/or an oxygen-carryingperfluorocarbon, as described in related U.S. Application PublicationNo. 2007/0190042, the disclosure of which is incorporated herein byreference in its entirety. In one embodiment, a method of preserving apopulation of cells, useful in the methods and compositions describedherein, comprises contacting said population of cells with a cellcollection composition comprising an inhibitor of apoptosis and anoxygen-carrying perfluorocarbon, wherein said inhibitor of apoptosis ispresent in an amount and for a time sufficient to reduce or preventapoptosis in the population of cells, as compared to a population ofcells not contacted with the inhibitor of apoptosis. In a specificembodiment, said inhibitor of apoptosis is a caspase inhibitor. Inanother specific embodiment, said inhibitor of apoptosis is a JNKinhibitor. In another specific embodiment, said JNK inhibitor does notmodulate differentiation or proliferation of said cells. In anotherembodiment, said cell collection composition comprises said inhibitor ofapoptosis and said oxygen-carrying perfluorocarbon in separate phases.In another embodiment, said cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon in anemulsion. In another embodiment, the cell collection compositionadditionally comprises an emulsifier, e.g., lecithin. In anotherembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 0° C. and about 25° C. at the time of contacting thecells. In another specific embodiment, said apoptosis inhibitor and saidperfluorocarbon are between about 2° C. and 10° C., or between about 2°C. and about 5° C., at the time of contacting the cells. In anotherspecific embodiment, said contacting is performed during transport ofsaid population of cells. In another specific embodiment, saidcontacting is performed during freezing and thawing of said populationof cells.

Populations of placental cells can be preserved, e.g., by a methodcomprising contacting said population of cells with an inhibitor ofapoptosis and an organ-preserving compound, wherein said inhibitor ofapoptosis is present in an amount and for a time sufficient to reduce orprevent apoptosis in the population of cells, as compared to apopulation of cells not contacted with the inhibitor of apoptosis. In aspecific embodiment, the organ-preserving compound is UW solution(described in U.S. Pat. No. 4,798,824; also known as ViaSpan; see alsoSouthard et al., Transplantation 49(2):251-257 (1990)) or a solutiondescribed in Stern et al., U.S. Pat. No. 5,552,267, the disclosures ofwhich are hereby incorporated by reference in their entireties. Inanother embodiment, said organ-preserving compound is hydroxyethylstarch, lactobionic acid, raffinose, or a combination thereof. Inanother embodiment, the cell collection composition additionallycomprises an oxygen-carrying perfluorocarbon, either in two phases or asan emulsion.

In another embodiment of the method, placental cells are contacted witha cell collection composition comprising an apoptosis inhibitor andoxygen-carrying perfluorocarbon, organ-preserving compound, orcombination thereof, during perfusion. In another embodiment, said cellsare contacted during a process of tissue disruption, e.g., enzymaticdigestion. In another embodiment, placental cells are contacted withsaid cell collection compound after collection by perfusion, or aftercollection by tissue disruption, e.g., enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, acell, or population of cells, is exposed to a hypoxic condition duringcollection, enrichment or isolation for less than six hours during saidpreservation, wherein a hypoxic condition is a concentration of oxygenthat is less than normal blood oxygen concentration. In another specificembodiment, said population of cells is exposed to said hypoxiccondition for less than two hours during said preservation. In anotherspecific embodiment, said population of cells is exposed to said hypoxiccondition for less than one hour, or less than thirty minutes, or is notexposed to a hypoxic condition, during collection, enrichment orisolation. In another specific embodiment, said population of cells isnot exposed to shear stress during collection, enrichment or isolation.

Placental cells can be cryopreserved, e.g., in cryopreservation mediumin small containers, e.g., ampoules. Suitable cryopreservation mediumincludes, but is not limited to, culture medium including, e.g., growthmedium, or cell freezing medium, for example commercially available cellfreezing medium, e.g., C2695, C2639 or C6039 (Sigma). Cryopreservationmedium preferably comprises DMSO (dimethylsulfoxide), at a concentrationof about 2% to about 15% (v/v), e.g., about 10% (v/v). Cryopreservationmedium may comprise additional agents, for example, methylcelluloseand/or glycerol. Placental cells are preferably cooled at about 1°C./min during cryopreservation. A preferred cryopreservation temperatureis about −80° C. to about −180° C., preferably about −125° C. to about−140° C. Cryopreserved cells can be transferred to liquid nitrogen priorto thawing for use. In some embodiments, for example, once the ampouleshave reached about −90° C., they are transferred to a liquid nitrogenstorage area. Cryopreservation can also be done using a controlled-ratefreezer. Cryopreserved cells preferably are thawed at a temperature ofabout 25° C. to about 40° C., preferably to a temperature of about 37°C.

4.7 Compositions Comprising Isolated Placental Cells

The placental cells described herein, e.g., at Section 4.4.2, can becombined with any physiologically-acceptable or medically-acceptablecompound, composition or device for use in the methods and compositionsdescribed herein. Compositions useful in the methods of treatmentprovided herein can comprise any one or more of the placental cellsdescribed herein (see Section 4.4.2, above). In certain embodiments, thecomposition is a pharmaceutically-acceptable composition, e.g., acomposition comprising placental cells in a pharmaceutically-acceptablecarrier. See Section 4.9.2, below.

In certain embodiments, a composition comprising the isolated placentalcells additionally comprises a matrix, e.g., a decellularized matrix ora synthetic matrix. In another specific embodiment, said matrix is athree-dimensional scaffold. In another specific embodiment, said matrixcomprises collagen, gelatin, laminin, fibronectin, pectin, ornithine, orvitronectin. In another ore specific embodiment, the matrix is anamniotic membrane or an amniotic membrane-derived biomaterial. Inanother specific embodiment, said matrix comprises an extracellularmembrane protein. In another specific embodiment, said matrix comprisesa synthetic compound. In another specific embodiment, said matrixcomprises a bioactive compound. In another specific embodiment, saidbioactive compound is a growth factor, cytokine, antibody, or organicmolecule of less than 5,000 daltons.

In another embodiment, a composition useful in the methods of treatmentprovided herein comprises medium conditioned by any of the foregoingplacental cells, or any of the foregoing placental cell populations.

4.7.1 Cryopreserved Isolated Placental Cells

The isolated placental cell populations useful in the methods andcompositions described herein can be preserved, for example,cryopreserved for later use. Methods for cryopreservation of cells, suchas stem cells, are well known in the art. Isolated placental cellpopulations can be prepared in a form that is easily administrable to anindividual, e.g., an isolated placental cell population that iscontained within a container that is suitable for medical use. Such acontainer can be, for example, a syringe, sterile plastic bag, flask,jar, or other container from which the isolated placental cellpopulation can be easily dispensed. For example, the container can be ablood bag or other plastic, medically-acceptable bag suitable for theintravenous administration of a liquid to a recipient. The container ispreferably one that allows for cryopreservation of the combined cellpopulation.

The cryopreserved isolated placental cell population can compriseisolated placental cell derived from a single donor, or from multipledonors. The isolated placental cell population can be completelyHLA-matched to an intended recipient, or partially or completelyHLA-mismatched.

Thus, in one embodiment, isolated placental cells can be used in themethods and described herein in the form of a composition comprising atissue culture plastic-adherent placental cell population in acontainer. In a specific embodiment, the isolated placental cells arecryopreserved. In another specific embodiment, the container is a bag,flask, or jar. In another specific embodiment, said bag is a sterileplastic bag. In another specific embodiment, said bag is suitable for,allows or facilitates intravenous administration of said isolatedplacental cell population, e.g., by intravenous infusion. The bag cancomprise multiple lumens or compartments that are interconnected toallow mixing of the isolated placental cells and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined cell population. Inanother specific embodiment, said isolated placental cell population iscontained within a physiologically-acceptable aqueous solution. Inanother specific embodiment, said physiologically-acceptable aqueoussolution is a 0.9% NaCl solution. In another specific embodiment, saidisolated placental cell population comprises placental cells that areHLA-matched to a recipient of said cell population. In another specificembodiment, said combined cell population comprises placental cells thatare at least partially HLA-mismatched to a recipient of said cellpopulation. In another specific embodiment, said isolated placentalcells are derived from a plurality of donors.

In certain embodiments, the isolated placental cells in the containerare isolated CD10⁺, CD34⁻, CD105⁺ placental cells, wherein said cellshave been cryopreserved, and are contained within a container. In aspecific embodiment, said CD10⁺, CD34⁻, CD105⁺ placental cells are alsoCD200⁺. In another specific embodiment, said CD10⁺, CD34⁻, CD105⁺,CD200⁺ placental cells are also CD45⁻ or CD90⁺. In another specificembodiment, said CD10⁺, CD34⁻, CD105⁺, CD200⁺ placental cells are alsoCD45⁻ and CD90⁺. In another specific embodiment, the CD34⁻, CD10⁺,CD105⁺ placental cells are additionally one or more of CD13⁺, CD29⁺,CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺),SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(dim), CD184/CXCR⁴⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, orProgrammed Death-1 Ligand (PDL1)⁺, or any combination thereof. Inanother specific embodiment, the CD34⁻, CD10⁺, CD105⁺ placental cellsare additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54/ICAM⁺,CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺,SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻, CD144/VE-cadherin^(dim),CD184/CXCR⁴⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻(VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, and Programmed Death-1Ligand (PDL1)⁺.

In certain other embodiments, the above-referenced isolated placentalcells are isolated CD200⁺, HLA-G⁻ placental cells, wherein said cellshave been cryopreserved, and are contained within a container. Inanother embodiment, the isolated placental cells are CD73⁺, CD105⁺,CD200⁺ cells that have been cryopreserved, and are contained within acontainer. In another embodiment, the isolated placental cells areCD200⁺, OCT-4⁺ stem cells that have been cryopreserved, and arecontained within a container. In another embodiment, the isolatedplacental cells are CD73⁺, CD105⁺ cells that have been cryopreserved,and are contained within a container, and wherein said isolatedplacental cells facilitate the formation of one or more embryoid-likebodies when cultured with a population of placental cells underconditions that allow for the formation of embryoid-like bodies. Inanother embodiment, the isolated placental cells are CD73⁺, CD105⁺,HLA-G⁻ cells that have been cryopreserved, and are contained within acontainer. In another embodiment, the isolated placental cells areOCT-4⁺ placental cells that have been cryopreserved, and are containedwithin a container, and wherein said cells facilitate the formation ofone or more embryoid-like bodies when cultured with a population ofplacental cells under conditions that allow for the formation ofembryoid-like bodies.

In another specific embodiment, the above-referenced isolated placentalcells are placental stem cells or placental multipotent cells that areCD34⁻, CD10⁺ and CD105⁺ as detected by flow cytometry (e.g., PDACs). Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placentalcells have the potential to differentiate into cells of a neuralphenotype, cells of an osteogenic phenotype, or cells of a chondrogenicphenotype. In another specific embodiment, the isolated CD34⁻, CD10⁺,CD105⁺ placental cells are additionally CD200⁺. In another specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cells areadditionally CD90⁺ or CD45⁻, as detected by flow cytometry. In anotherspecific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ placental cellsare additionally CD90⁺ or CD45⁻, as detected by flow cytometry. Inanother specific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ placentalcells are additionally CD90⁺ or CD45⁻, as detected by flow cytometry. Inanother specific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺ cells areadditionally CD90⁺ and CD45⁻, as detected by flow cytometry. In anotherspecific embodiment, the CD34⁻, CD10⁺, CD105⁺, CD200⁺, CD90⁺, CD45⁻cells are additionally CD80⁻ and CD86⁻, as detected by flow cytometry.In another specific embodiment, the CD34⁻, CD10⁺, CD105⁺ cells areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, CD80⁻, CD86⁻,SH3⁺ or SH4⁺. In another specific embodiment, the cells are additionallyCD44⁺. In a specific embodiment of any of the isolated CD34⁻, CD10⁺,CD105⁺ placental cells above, the cells are additionally one or more ofCD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻, HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, and/or PDL1⁺.

In a specific embodiment of any of the foregoing cryopreserved isolatedplacental cells, said container is a bag. In various specificembodiments, said container comprises about, at least, or at most 1×10⁶said isolated placental cells, 5×10⁶ said isolated placental cells,1×10⁷ said isolated placental cells, 5×10⁷ said isolated placentalcells, 1×10⁸ said isolated placental cells, 5×10⁸ said isolatedplacental cells, 1×10⁹ said isolated placental cells, 5×10⁹ saidisolated placental cells, 1×10¹⁰ said isolated placental cells, or1×10¹⁰ said isolated placental cells. In other specific embodiments ofany of the foregoing cryopreserved populations, said isolated placentalcells have been passaged about, at least, or no more than 5 times, nomore than 10 times, no more than 15 times, or no more than 20 times. Inanother specific embodiment of any of the foregoing cryopreservedisolated placental cells, said isolated placental cells have beenexpanded within said container.

4.7.2 Genetically Engineered Placental Cells

Further provided herein are placental cells, e.g., any of the placentalmultipotent cells or placental cells described in Section 4.2.2, above,or pharmaceutical compositions comprising such placental cells, whereinthe placental cells have been genetically engineered to producerecombinant or exogenous cytokines.

Methods for genetically engineering cells, for example with retroviralvectors, adenoviral vectors, adeno-associated viral vectors,polyethylene glycol, or other methods known to those skilled in the art,can be used. These include using expression vectors which transport andexpress nucleic acid molecules in the cells. (See Geoddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). Vector DNA can be introduced into prokaryotic oreukaryotic cells via conventional transformation or transfectiontechniques. Suitable methods for transforming or transfecting host cellscan be found in Sambrook et al. Molecular Cloning: A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory press (1989), and otherlaboratory textbooks.

Placental cells, e.g., the PDACs described in Section 4.2, above, can begenetically modified by introducing DNA or RNA into the cell, e.g., DNAor RNA encoding a protein of interest, by methods including viraltransfer, including the use of DNA or RNA viral vectors, such asretroviruses (including lentiviruses), Simian virus 40 (SV40),adenovirus, Sindbis virus, and bovine papillomavirus for example;chemical transfer, including calcium phosphate transfection and DEAEdextran transfection methods; membrane fusion transfer, using DNA-loadedmembrane vesicles such as liposomes, red blood cell ghosts, andprotoplasts, for example; or physical transfer techniques, such asmicroinjection, electroporation, or naked DNA transfer. The placentalcells can be genetically altered by insertion of exogenous DNA, or bysubstitution of a segment of the cellular genome with exogenous DNA.Insertion of exogenous DNA sequence(s) can be accomplished, e.g., byhomologous recombination or by viral integration into the host cellgenome, or by incorporating the DNA into the cell, particularly into itsnucleus, using a plasmid expression vector and a nuclear localizationsequence. The DNA can comprise one or more promoters that allow positiveor negative induction of expression of the protein of interest usingcertain chemicals/drugs, e.g., tetracycline; the promoters can, in otherembodiments, be constitutive.

Calcium phosphate transfection can be used to introduce, e.g., plasmidDNA containing a polynucleotide sequence encoding the protein ofinterest, into a cell. In certain embodiments, DNA is combined with asolution of calcium chloride, then added to a phosphate-bufferedsolution. Once a precipitate has formed, the solution is added directlyto cultured cells. Treatment with DMSO or glycerol can be used toimprove transfection efficiency, and levels of stable transfectants canbe improved using bis-hydroxyethylamino ethanesulfonate (BES). Calciumphosphate transfection systems are commercially available (e.g.,PROFECTION®, Promega Corp., Madison, Wis.). DEAF-dextran transfectionmay also be used.

Isolated placental cells may also be genetically engineered bymicroinjection. In certain embodiments, a glass micropipette is guidedinto the nucleus of cells under a light microscope to inject DNA or RNA.

Placental cells can also be genetically modified using electroporation.In certain embodiments, DNA or RNA is added to a suspension of culturedcells, and the DNA/RNA-cell suspension is placed between two electrodesand subjected to an electrical pulse, causing a transient permeabilityin the cell's outer membrane that is manifested by the appearance ofpores across the membrane.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, optionally including dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC),e.g., LIPOFECTIN® (Life Technologies, Inc.). Othercommercially-available delivery systems include EFFECTENE™ (Qiagen),DOTAP (Roche Molecular Biochemicals), FUGENE 6™. (Roche MolecularBiochemicals), and TRANSFECTAM® (Promega).

Viral vectors can be used to genetically alter placental cells bydelivery of, e.g., target genes, polynucleotides, antisense molecules,or ribozyme sequences into the cells. Retroviral vectors are effectivefor transducing rapidly-dividing cells, although a number of retroviralvectors have been developed to effectively transfer DNA intonon-dividing cells as well. Packaging cell lines for retroviral vectorsare known to those of skill in the art. In certain embodiments, aretroviral DNA vector contains two retroviral LTRs such that a first LTRis located 5′ to the SV40 promoter, which is operationally linked to thetarget gene sequence cloned into a multicloning site, followed by a 3′second LTR. Once formed, the retroviral DNA vector is transferred into apackaging cell line using calcium phosphate-mediated transfection, aspreviously described. Following approximately 48 hours of virusproduction, the viral vector, now containing the target gene sequence,is harvested. Methods of transfecting cells using lentiviral vectors,recombinant herpes viruses, adenoviral vectors, or alphavirus vectorsare known in the art.

Successful transfection or transduction of target cells can bedemonstrated using genetic markers, in a technique that is known tothose of skill in the art. The green fluorescent protein of Aequoreavictoria, for example, has been shown to be an effective marker foridentifying and tracking genetically modified hematopoietic cells.Alternative selectable markers include the β-Gal gene, truncated nervegrowth factor receptor, or drug selectable markers (including but notlimited to NEO, MTX, or hygromycin).

4.7.3 Pharmaceutical Compositions

Populations of isolated placental cells, e.g., PDACs, or populations ofcells comprising the isolated placental cells, can be formulated intopharmaceutical compositions for use in vivo, e.g., in the methods oftreatment provided herein. Such pharmaceutical compositions comprise apopulation of isolated placental cells, or a population of cellscomprising isolated placental cells, in a pharmaceutically-acceptablecarrier, e.g., a saline solution or other acceptedphysiologically-acceptable solution for in vivo administration.Pharmaceutical compositions comprising the isolated placental cellsdescribed herein can comprise any, or any combination, of the isolatedplacental cell populations, or isolated placental cells, describedelsewhere herein. The pharmaceutical compositions can comprise fetal,maternal, or both fetal and maternal isolated placental cells. Thepharmaceutical compositions provided herein can further compriseisolated placental cells obtained from a single individual or placenta,or from a plurality of individuals or placentae.

The pharmaceutical compositions provided herein can comprise any numberof isolated placental cells. For example, a single unit dose of isolatedplacental cells can comprise, in various embodiments, about, at least,or no more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹°, 5×10¹°, 1×10¹¹ or more isolated placental cells.

The pharmaceutical compositions provided herein comprise populations ofcells that comprise 50% viable cells or more (that is, at least 50% ofthe cells in the population are functional or living). Preferably, atleast 60% of the cells in the population are viable. More preferably, atleast 70%, 80%, 90%, 95%, or 99% of the cells in the population in thepharmaceutical composition are viable.

The pharmaceutical compositions provided herein can comprise one or morecompounds that, e.g., facilitate engraftment (e.g., anti-T-cell receptorantibodies, an immunosuppressant, or the like); stabilizers such asalbumin, dextran 40, gelatin, hydroxyethyl starch, plasmalyte, and thelike.

When formulated as an injectable solution, in one embodiment, thepharmaceutical composition comprises about 1% to 1.5% HSA and about 2.5%dextran. In a preferred embodiment, the pharmaceutical compositioncomprises from about 5×10⁶ cells per milliliter to about 2×10⁷ cells permilliliter in a solution comprising 5% HSA and 10% dextran, optionallycomprising an immunosuppressant, e.g., cyclosporine A at, e.g., 10mg/kg.

In other embodiments, the pharmaceutical composition, e.g., a solution,comprises a plurality of cells, e.g., isolated placental cells, forexample, placental stem cells or placental multipotent cells, whereinsaid pharmaceutical composition comprises between about 1.0±0.3×10⁶cells per milliliter to about 5.0±1.5×10⁶ cells per milliliter. In otherembodiments, the pharmaceutical composition comprises between about1.5×10⁶ cells per milliliter to about 3.75×10⁶ cells per milliliter. Inother embodiments, the pharmaceutical composition comprises betweenabout 1×10⁶ cells/mL to about 50×10⁶ cells/mL, about 1×10⁶ cells/mL toabout 40×10⁶ cells/mL, about 1×10⁶ cells/mL to about 30×10⁶ cells/mL,about 1×10⁶ cells/mL to about 20×10⁶ cells/mL, about 1×10⁶ cells/mL toabout 15×10⁶ cells/mL, or about 1×10⁶ cells/mL to about 10×10⁶ cells/mL.In certain embodiments, the pharmaceutical composition comprises novisible cell clumps (i.e., no macro cell clumps), or substantially nosuch visible clumps. As used herein, “macro cell clumps” means anaggregation of cells visible without magnification, e.g., visible to thenaked eye, and generally refers to a cell aggregation larger than about150 microns In some embodiments, the pharmaceutical compositioncomprises about 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%,7.0%, 7.5% 8.0%, 8.5%, 9.0%, 9.5% or 10% dextran, e.g., dextran-40. In aspecific embodiment, said composition comprises about 7.5% to about 9%dextran-40. In a specific embodiment, said composition comprises about5.5% dextran-40. In certain embodiments, the pharmaceutical compositioncomprises from about 1% to about 15% human serum albumin (HSA). Inspecific embodiments, the pharmaceutical composition comprises about 1%,2%, 3%, 4%, 5%, 65, 75, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% HSA. In aspecific embodiment, said cells have been cryopreserved and thawed. Inanother specific embodiment, said cells have been filtered through a 70μM to 100 μM filter. In another specific embodiment, said compositioncomprises no visible cell clumps. In another specific embodiment, saidcomposition comprises fewer than about 200 cell clumps per 10⁶ cells,wherein said cell clumps are visible only under a microscope, e.g., alight microscope. In another specific embodiment, said compositioncomprises fewer than about 150 cell clumps per 10⁶ cells, wherein saidcell clumps are visible only under a microscope, e.g., a lightmicroscope. In another specific embodiment, said composition comprisesfewer than about 100 cell clumps per 10⁶ cells, wherein said cell clumpsare visible only under a microscope, e.g., a light microscope.

In a specific embodiment, the pharmaceutical composition comprises about1.0±0.3×10⁶ cells per milliliter, about 5.5% dextran-40 (w/v), about 10%HSA (w/v), and about 5% DMSO (v/v).

In other embodiments, the pharmaceutical composition comprises aplurality of cells, e.g., a plurality of isolated placental cells in asolution comprising 10% dextran-40, wherein the pharmaceuticalcomposition comprises between about 1.0±0.3×10⁶ cells per milliliter toabout 5.0±1.5×10⁶ cells per milliliter, and wherein said compositioncomprises no cell clumps visible with the unaided eye (i.e., comprisesno macro cell clumps). In some embodiments, the pharmaceuticalcomposition comprises between about 1.5×10⁶ cells per milliliter toabout 3.75×10⁶ cells per milliliter. In a specific embodiment, saidcells have been cryopreserved and thawed. In another specificembodiment, said cells have been filtered through a 70 μM to 100 μMfilter. In another specific embodiment, said composition comprises fewerthan about 200 micro cell clumps (that is, cell clumps visible only withmagnification) per 10⁶ cells. In another specific embodiment, thepharmaceutical composition comprises fewer than about 150 micro cellclumps per 10⁶ cells. In another specific embodiment, the pharmaceuticalcomposition comprises fewer than about 100 micro cell clumps per 10⁶cells. In another specific embodiment, the pharmaceutical compositioncomprises less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, or 2% DMSO, or less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, or 0.1% DMSO.

Further provided herein are compositions comprising cells, wherein saidcompositions are produced by one of the methods disclosed herein. Forexample, in one embodiment, the pharmaceutical composition comprisescells, wherein the pharmaceutical composition is produced by a methodcomprising filtering a solution comprising placental cells, e.g.,placental stem cells or placental multipotent cells, to form a filteredcell-containing solution; diluting the filtered cell-containing solutionwith a first solution to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1to 20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter, e.g., priorto cryopreservation; and diluting the resulting filtered cell-containingsolution with a second solution comprising dextran, but not comprisinghuman serum albumin (HSA) to produce said composition. In certainembodiments, said diluting is to no more than about 15×10⁶ cells permilliliter. In certain embodiments, said diluting is to no more thanabout 10±3×10⁶ cells per milliliter. In certain embodiments, saiddiluting is to no more than about 7.5×10⁶ cells per milliliter. In othercertain embodiments, if the filtered cell-containing solution, prior tothe dilution, comprises less than about 15×10⁶ cells per milliliter,filtration is optional. In other certain embodiments, if the filteredcell-containing solution, prior to the dilution, comprises less thanabout 10±3×10⁶ cells per milliliter, filtration is optional. In othercertain embodiments, if the filtered cell-containing solution, prior tothe dilution, comprises less than about 7.5×10⁶ cells per milliliter,filtration is optional.

In a specific embodiment, the cells are cryopreserved between saiddiluting with a first dilution solution and said diluting with saidsecond dilution solution. In another specific embodiment, the firstdilution solution comprises dextran and HSA. The dextran in the firstdilution solution or second dilution solution can be dextran of anymolecular weight, e.g., dextran having a molecular weight of from about10 kDa to about 150 kDa. In some embodiments, said dextran in said firstdilution solution or said second solution is about 2.5%, 3.0%, 3.5%,4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%, 8.5%, 9.0%, 9.5% or10% dextran. In another specific embodiment, the dextran in said firstdilution solution or said second dilution solution is dextran-40. Inanother specific embodiment, the dextran in said first dilution solutionand said second dilution solution is dextran-40. In another specificembodiment, said dextran-40 in said first dilution solution is 5.0%dextran-40. In another specific embodiment, said dextran-40 in saidfirst dilution solution is 5.5% dextran-40. In another specificembodiment, said dextran-40 in said second dilution solution is 10%dextran-40. In another specific embodiment, said HSA in said solutioncomprising HSA is 1 to 15% HSA. In another specific embodiment, said HSAin said solution comprising HSA is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14% or 15 HSA. In another specific embodiment,said HSA in said solution comprising HSA is 10% HSA. In another specificembodiment, said first dilution solution comprises HSA. In anotherspecific embodiment, said HSA in said first dilution solution is 10%HSA. In another specific embodiment, said first dilution solutioncomprises a cryoprotectant. In another specific embodiment, saidcryoprotectant is DMSO. In another specific embodiment, said dextran-40in said second dilution solution is about 10% dextran-40. In anotherspecific embodiment, said composition comprising cells comprises about7.5% to about 9% dextran. In another specific embodiment, thepharmaceutical composition comprises from about 1.0±0.3×10⁶ cells permilliliter to about 5.0±1.5×10⁶ cells per milliliter. In anotherspecific embodiment, the pharmaceutical composition comprises from about1.5×10⁶ cells per milliliter to about 3.75×10⁶ cells per milliliter.

In another embodiment, the pharmaceutical composition is made by amethod comprising (a) filtering a cell-containing solution comprisingplacental cells, e.g., placental stem cells or placental multipotentcells, prior to cryopreservation to produce a filtered cell-containingsolution; (b) cryopreserving the cells in the filtered cell-containingsolution at about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to 20×10⁶, 1to 15×10⁶, or 1 to 10×10⁶ cells per milliliter; (c) thawing the cells;and (d) diluting the filtered cell-containing solution about 1:1 toabout 1:11 (v/v) with a dextran-40 solution. In certain embodiments, ifthe number of cells is less than about 10±3×10⁶ cells per milliliterprior to step (a), filtration is optional. In another specificembodiment, the cells in step (b) are cryopreserved at about 10±3×10⁶cells per milliliter. In another specific embodiment, the cells in step(b) are cryopreserved in a solution comprising about 5% to about 10%dextran-40 and HSA. In certain embodiments, said diluting in step (b) isto no more than about 15×10⁶ cells per milliliter.

In another embodiment, the pharmaceutical composition is made by amethod comprising: (a) suspending placental cells, e.g., placental stemcells or placental multipotent cells, in a 5.5% dextran-40 solution thatcomprises 10% HSA to form a cell-containing solution; (b) filtering thecell-containing solution through a 70 μM filter; (c) diluting thecell-containing solution with a solution comprising 5.5% dextran-40, 10%HSA, and 5% DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cellsper milliliter; (d)cryopreserving the cells; (e) thawing the cells; and (f) diluting thecell-containing solution 1:1 to 1:11 (v/v) with 10% dextran-40. Incertain embodiments, said diluting in step (c) is to no more than about15×10⁶ cells per milliliter. In certain embodiments, said diluting instep (c) is to no more than about 10±3×10⁶ cells/mL. In certainembodiments, said diluting in step (c) is to no more than about 7.5×10⁶cells/mL.

In another embodiment, the composition comprising cells is made by amethod comprising: (a) centrifuging a plurality of cells to collect thecells; (b) resuspending the cells in 5.5% dextran-40; (c) centrifugingthe cells to collect the cells; (d) resuspending the cells in a 5.5%dextran-40 solution that comprises 10% HSA; (e) filtering the cellsthrough a 70 μM filter; (f) diluting the cells in 5.5% dextran-40, 10%HSA, and 5% DMSO to about 1 to 50×10⁶, 1 to 40×10⁶, 1 to 30×10⁶, 1 to20×10⁶, 1 to 15×10⁶, or 1 to 10×10⁶ cells per milliliter; (g)cryopreserving the cells; (h) thawing the cells; and (i) diluting thecells 1:1 to 1:11 (v/v) with 10% dextran-40. In certain embodiments,said diluting in step (f) is to no more than about 15×10⁶ cells permilliliter. In certain embodiments, said diluting in step (f) is to nomore than about 10±3×10⁶ cells/mL. In certain embodiments, said dilutingin step (f) is to no more than about 7.5×10⁶ cells/mL. In other certainembodiments, if the number of cells is less than about 10±3×10⁶ cellsper milliliter, filtration is optional.

The compositions, e.g., pharmaceutical compositions comprising theisolated placental cells, described herein can comprise any of theisolated placental cells described herein.

Other injectable formulations, suitable for the administration ofcellular products, may be used.

In one embodiment, the pharmaceutical composition comprises isolatedplacental cells that are substantially, or completely, non-maternal inorigin, that is, have the fetal genotype; e.g., at least about 90%, 95%,98%, 99% or about 100% are non-maternal in origin. For example, in oneembodiment a pharmaceutical composition comprises a population ofisolated placental cells that are CD200⁺ and HLA-G⁻; CD73⁺, CD105⁺, andCD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁻; CD73⁺ and CD105⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said population of isolatedplacental cell when said population of placental cells is cultured underconditions that allow the formation of an embryoid-like body; or OCT-4⁺and facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said population of isolatedplacental cell when said population of placental cells is cultured underconditions that allow the formation, of an embryoid-like body; or acombination of the foregoing, wherein at least 70%, 80%, 90%, 95% or 99%of said isolated placental cells are non-maternal in origin. In anotherembodiment, a pharmaceutical composition comprises a population ofisolated placental cells that are CD10⁺, CD105⁺ and CD34⁻; CD10⁺,CD105⁺, CD200⁺ and CD34⁻; CD10⁺, CD105⁺, CD200⁺, CD34⁻ and at least oneof CD90⁺ or CD45⁻; CD10⁺, CD90⁺, CD105⁺, CD200⁺, CD34⁻ and CD45⁻; CD10⁺,CD90⁺, CD105⁺, CD200⁺, CD34⁻ and CD45⁻; CD200⁺ and HLA-G⁻; CD73⁺,CD105⁺, and CD200⁺; CD200⁺ and OCT-4⁺; CD73⁺, CD105⁺ and HLA-G⁻; CD73⁺and CD105⁺ and facilitate the formation of one or more embryoid-likebodies in a population of placental cells comprising said isolatedplacental cells when said population of placental cells is culturedunder conditions that allow the formation of an embryoid-like body;OCT-4⁺ and facilitate the formation of one or more embryoid-like bodiesin a population of placental cells comprising said isolated placentalcells when said population of placental cells is cultured underconditions that allow the formation of an embryoid-like body; or one ormore of CD117⁻, CD133⁻, KDR⁻, CD80⁻, CD86⁻, HLA-A,B,C⁺, HLA-DP,DQ,DR⁻and/or PDL1⁺; or a combination of the foregoing, wherein at least 70%,80%, 90%, 95% or 99% of said isolated placental cells are non-maternalin origin. In a specific embodiment, the pharmaceutical compositionadditionally comprises a stem cell that is not obtained from a placenta.

Isolated placental cells in the compositions, e.g., pharmaceuticalcompositions, provided herein, can comprise placental cells derived froma single donor, or from multiple donors. The isolated placental cellscan be completely HLA-matched to an intended recipient, or partially orcompletely HLA-mismatched.

4.7.4 Matrices Comprising Isolated Placental Cells

Further provided herein are compositions comprising matrices, hydrogels,scaffolds, and the like that comprise a placental cell, or a populationof isolated placental cells. Such compositions can be used in the placeof, or in addition to, cells in liquid suspension.

The isolated placental cells described herein can be seeded onto anatural matrix, e.g., a placental biomaterial such as an amnioticmembrane material. Such an amniotic membrane material can be, e.g.,amniotic membrane dissected directly from a mammalian placenta; fixed orheat-treated amniotic membrane, substantially dry (i.e., <20% H₂O)amniotic membrane, chorionic membrane, substantially dry chorionicmembrane, substantially dry amniotic and chorionic membrane, and thelike. Preferred placental biomaterials on which isolated placental cellscan be seeded are described in Hariri, U.S. Application Publication No.2004/0048796, the disclosure of which is incorporated herein byreference in its entirety.

The isolated placental cells described herein can be suspended in ahydrogel solution suitable for, e.g., injection. Suitable hydrogels forsuch compositions include self-assembling peptides, such as RAD16. Inone embodiment, a hydrogel solution comprising the cells can be allowedto harden, for instance in a mold, to form a matrix having cellsdispersed therein for implantation. Isolated placental cells in such amatrix can also be cultured so that the cells are mitotically expandedprior to implantation. The hydrogel is, e.g., an organic polymer(natural or synthetic) that is cross-linked via covalent, ionic, orhydrogen bonds to create a three-dimensional open-lattice structure thatentraps water molecules to form a gel. Hydrogel-forming materialsinclude polysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are crosslinked ionically, orblock polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are crosslinked by temperature or pH, respectively. Insome embodiments, the hydrogel or matrix is biodegradable.

In some embodiments, the formulation comprises an in situ polymerizablegel (see., e.g., U.S. Patent Application Publication 2002/0022676, thedisclosure of which is incorporated herein by reference in its entirety;Anseth et al., J. Control Release, 78(1-3):199-209 (2002); Wang et al.,Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

The isolated placental cells described herein or co-cultures thereof canbe seeded onto a three-dimensional framework or scaffold and implantedin vivo. Such a framework can be implanted in combination with any oneor more growth factors, cells, drugs or other components that, e.g.,stimulate tissue formation.

Examples of scaffolds that can be used include nonwoven mats, porousfoams, or self assembling peptides. Nonwoven mats can be formed usingfibers comprised of a synthetic absorbable copolymer of glycolic andlactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.).Foams, composed of, e.g., poly(ε-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be used asscaffolds.

In another embodiment, isolated placental cells can be seeded onto, orcontacted with, a felt, which can be, e.g., composed of a multifilamentyarn made from a bioabsorbable material such as PGA, PLA, PCL copolymersor blends, or hyaluronic acid.

The isolated placental cells provided herein can, in another embodiment,be seeded onto foam scaffolds that may be composite structures. Suchfoam scaffolds can be molded into a useful shape, such as that of aportion of a specific structure in the body to be repaired, replaced oraugmented. In some embodiments, the framework is treated, e.g., with0.1M acetic acid followed by incubation in polylysine, PBS, and/orcollagen, prior to inoculation of the cells in order to enhance cellattachment. External surfaces of a matrix may be modified to improve theattachment or growth of cells and differentiation of tissue, such as byplasma-coating the matrix, or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), acellular matrix, and/or other materials such as, but not limited to,gelatin, alginates, agar, agarose, and plant gums, and the like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with isolatedplacental cells.

The placental cells (e.g., PDACs) provided herein can also be seededonto, or contacted with, a physiologically-acceptable ceramic materialincluding, but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-,and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materialscurrently commercially available include SURGIBONE® (CanMedica Corp.,Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys,AG, Bettlach, Switzerland), and mineralized collagen bone graftingproducts such as HEALOS™ (DePuy, Inc., Raynham, Mass.) and VITOSS®,RHAKOSS™, and CORTOSS® (Orthovita, Malvern, Pa.). The framework can be amixture, blend or composite of natural and/or synthetic materials.

In one embodiment, the isolated placental cells are seeded onto, orcontacted with, a suitable scaffold at about 0.5×10⁶ to about 8×10⁶cells/mL.

4.8 Immortalized Placental Cell Lines

Mammalian placental cells, e.g., PDACs, can be conditionallyimmortalized by transfection with any suitable vector containing agrowth-promoting gene, that is, a gene encoding a protein that, underappropriate conditions, promotes growth of the transfected cell, suchthat the production and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1aadenovirus or E7 protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. in one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV*−1), a minimal promoter from human cytomegalovirus fused totet operator sequences. tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed within the present methods. Cells carrying neo^(R) may beselected by means known to those of ordinary skill in the art, such asthe addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized placental cell lines can be passagedusing standard techniques, such as by trypsinization, when 80-95%confluent. Up to approximately the twentieth passage, it is, in someembodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman placental cell line prepared as described above. In general, suchclonal cell lines may be isolated using standard techniques, such as bylimit dilution or using cloning rings, and expanded. Clonal cell linesmay generally be fed and passaged as described above.

Conditionally-immortalized human placental cell lines, which may, butneed not, be clonal, may generally be induced to differentiate bysuppressing the production and/or activity of the growth-promotingprotein under culture conditions that facilitate differentiation. Forexample, if the gene encoding the growth-promoting protein is under thecontrol of an externally-regulatable promoter, the conditions, e.g.,temperature or composition of medium, may be modified to suppresstranscription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

4.9 Kits

In another aspect, provided herein are kits, suitable for the treatmentof an individual who has sarcoidosis or a sarcoidosis-related disease ordisorder, comprising, in a container separate from remaining kitcontents, tissue culture plastic adherent multipotent placental cells,e.g., placental stem cells or placental multipotent cells, e.g., thecells described in Section 4.2, above (PDACs), and instructions for use.Ideally the kit can be used in the field, for example in a physician'soffice, or by an emergency care provider to be applied to a patientdiagnosed as having sarcoidosis or a sarcoidosis-related disease ordisorder. Preferably, the placental cells are provided in apharmaceutically-acceptable solution, e.g., a solution suitable forintralesional administration or a solution suitable for intravenousadministration. In certain embodiments, the placental stem cells orplacental multipotent cells are any of the CD10⁺, CD34⁻, CD105⁺placental cells described herein, e.g., CD10⁺, CD34⁻, CD105⁺, CD200⁺placental cells or CD10⁺, CD34⁻, CD45⁻, CD90⁺, CD105⁺, CD200⁺ placentalcells.

In certain embodiments, the kits comprise one or more components thatfacilitate delivery of the placental cells to the individual. Forexample, in certain embodiments, the kit comprises components thatfacilitate intralesional delivery of the placental cells to theindividual. In such embodiments, the kit can comprise, e.g., syringesand needles suitable for delivery of cells to the individual, and thelike. In such embodiments, the placental cells may be contained in thekit in a bag, or in one or more vials. In certain other embodiments, thekit comprises components that facilitate intravenous or intra-arterialdelivery of the placental cells to the individual. In such embodiments,the placental cells may be contained, e.g., within a bottle or bag (forexample, a blood bag or similar bag able to contain up to about 1.5 Lsolution comprising the cells), and the kit additionally comprisestubing and needles suitable for the delivery of cells to the individual.

Additionally, the kit may comprise one or more compounds that reducepain or inflammation in the individual (e.g., an analgesic, steroidal ornon-steroidal anti-inflammatory compound, or the like. The kit may alsocomprise an antibacterial or antiviral compound (e.g., one or moreantibiotics), a compound to reduce anxiety in the individual (e.g.,alaprazolam), a compound that reduces an immune response in theindividual (e.g., cyclosporine A), an antihistamine (diphenhydramine,loratadine, desloratadine, quetiapine, fexofenadine, cetirizine,promethazine, chlorpheniramine, levocetirizine, cimetidine, famotidine,ranitidine, nizatidine, roxatidine, lafutidine, or the like).

Additionally, the kit can comprise disposables, e.g., sterile wipes,disposable paper goods, gloves, or the like, which facilitatepreparation of the individual for delivery, or which reduce thelikelihood of infection in the individual as a result of theadministration of the placental cells.

5. EXAMPLES 5.1 Example 1 Treatment of Systemic Sarcoidosis UsingPlacental Stem Cells

A male patient in his middle '40s reports symptoms of weakness,shortness of breath, exertional dyspnea, chest pain, moderate fever andankle and knee discomfort, and undergoes physical examination. The chestX-ray and CT scans reveal bilateral hilar and mediastinallymphadenopathy, parenchymal lesions in the right lung and a numbers ofnodules. Systemic sarcoidosis and infectious arthritis are diagnosed.The individual is administered 1×10⁹ to 5×10⁹ CD10⁺, CD34⁻, CD105⁺,CD200⁺ PDACs intravenously in 450 mL 0.9% saline, and is monitoredbiweekly for the next three months. Therapeutic effectiveness isestablished if any of the symptoms described above improve during themonitoring period.

5.2 Example 2 Treatment of Cardiac Sarcoidosis Using Placental StemCells

An individual presents with breathlessness, swelling of the legs andankles, and irregular heartbeats. After excluding other causes, and witha confirmatory electrocardiogram, a diagnosis of cardiac sarcoidosis yis made. After stabilization of the individual with nitroglycerin andstreptokinase, the individual is administered 1×10⁸ to 5×10⁸ CD10⁺,CD34⁻, CD105⁺, CD200⁺ PDACs in 0.9% saline directly to the affected areausing a cardiac syringe with local anesthetic. The individual ismonitored on an emergency basis for the next 72 hours. The individual isfurther monitored over the next three months port-treatment byelectrocardiogram and/or dye visualization techniques. Therapeuticeffectiveness is established if any of the symptoms described aboveimprove during the monitoring period.

5.3 Example 3 Treatment of Cutaneous Sarcoidosis Using Placental StemCells

An individual presents with complaints of nodules developing in thelower legs. A shave biopsy is performed and histologic examinationreveals granulomatous inflammation with polarizable foreign material.After excluding other causes, a diagnosis of cutaneous sarcoidosis ismade. The individual is administered 1×10⁸ to 5×10⁸ CD10⁺, CD34⁻,CD105⁺, CD200⁺ PDACs in 0.9% saline directly to the skin lesions. Theindividual is monitored over the next three months for changes in theskin nodules. Therapeutic effectiveness is established for theindividual if any of these sings show improvement during the monitoringperiod.

5.4 Example 4 Treatment of Neurosarcoidosis Using Placental Stem Cells

A 50-year-old individual exhibits symptoms of leg numbness and muscleweakness. Imaging modalities shows an irregular contour and a mass-likelesion in the spinal cord. A biopsy reveal fibrosis and non-caseatinggranulomata in the spinal cord. A diagnosis of neurosarcoidosis is made.The individual is administered 1×10⁹ to 5×10⁹ CD10⁺, CD34⁻, CD105⁺,CD200⁺ PDACs intrathecally in 450 mL 0.9% saline, and is monitoredbiweekly for the next three months. Therapeutic effectiveness isestablished if any of the symptoms described above improve during themonitoring period.

EQUIVALENTS

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of thesubject matter provided herein, in addition to those described, willbecome apparent to those skilled in the art from the foregoingdescription and accompanying figures. Such modifications are intended tofall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1. A method of treating an individual having sarcoidosis comprisingadministering to the individual a therapeutically effective amount ofplacental stem cells, wherein the therapeutically effective amount is anamount sufficient to cause a detectable improvement in one or moresymptoms of said sarcoidosis, and wherein said sarcoidosis is notpulmonary sarcoidosis.
 2. The method of claim 1, wherein said placentalstem cells are CD10⁺, CD34⁻, CD105⁺, CD200⁺ placental stem cells.
 3. Themethod of claim 2, wherein said placental stem cells are additionallyCD45⁻ or CD90⁺.
 4. The method of claim 2, wherein said placental stemcells are additionally one or more of CD38⁻, CD45⁻, CD80⁻, CD86⁻,CD133⁻, HLA-DR,DP,DQ⁻, SSEA3⁻, SSEA4⁻, CD29⁺, CD44⁺, CD73⁺, CD90⁺,HLA-A,B,C⁺, PDL1⁺, ABC-p⁺, or OCT-4⁺.
 5. The method of claim 2, whereinsaid placental stem cells are additionally one or more of CD29⁺, CD38⁻,CD44⁺, CD54⁺, SH3⁺ or SH4⁺.
 6. The method of claim 2, wherein saidplacental stem cells are additionally CD44⁺.
 7. The method of claim 1,wherein said placental stem cells express CD200 and do not expressHLA-G, or express CD73. CD105, and CD200, or express CD200 and OCT-4, orexpress CD73 and CD105 and do not express HLA-G, or express CD73 andCD105 and facilitate the formation of one or more embryoid-like bodiesin a population of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body, or express OCT-4 and facilitate the formation ofone or more embryoid-like bodies in a population of placental cellscomprising said stem cell when said population is cultured underconditions that allow for the formation of an embryoid-like body. 8-23.(canceled)
 24. The method of claim 1, comprising administering a secondtherapeutic agent to said individual.
 25. The method of claim 24,wherein said second therapeutic agent is an anti-inflammatory agent, asteroid, an immunosuppressant compound, or an antibiotic.